Self-assembling escrt recruiting domains (ERD) and methods of use thereof

Abstract

Disclosed herein are polynucleotides that encode fusion proteins, wherein the fusion proteins comprise an antigenic polypeptide and one or more non-human endosomal sorting complex required for transport-recruiting domains (ERDs). Also provided herein are related compositions, methods of making, and methods of using. In particular, these fusion proteins are useful to produce enveloped virus-like particles (eVLPs) for use in the prevention and treatment of diseases or disorders.

Description

SELF-ASSEMBLING ESCRT RECRUITING DOMAINS (ERD) AND METHODS OFUSE THEREOFRELATED APPLICATIONS

[0001] This application claims the priority and benefits of U.S. Provisional Application No. 63 / 653,204, filed May 29, 2024 and U.S. Provisional Application No. 63 / 778,296, filed March 26, 2025, 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_01 l_02WO_SeqList_ST26.xml; Size: 437,424 bytes; and Date of Creation: May 29, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0004] The Endosomal Sorting Complex Required for Transport (ESCRT) is a component of cellular machinery facilitating the transit of proteins and cargo within cells. Notably, it plays a role in shuttling proteins from the endoplasmic reticulum (ER) to the cell surface, as well as in the generation of extracellular vesicles, which can originate either within the ER, or at the cell surface before being extruded from the cell.

[0005] Many viruses, such as human immunodeficiency virus (HIV), Equine Infectious Anemia Virus (EIAV), and Mason-Pfizer monkey virus (MPMV), exploit ESCRT machinery to facilitate their budding from host cells. Vaccines containing human derived ESCRT recruiting domains (ERDs) may show promise in enhancing immunogenicity by inducing antigen expression on cell surfaces and promoting the formation of extracellular vesicles presenting antigens, known as enveloped virus-like particles (eVLPs). However, there is a need to increase the potency of these approaches, and there are concerns about incorporating sequences of humanorigin in vaccines, specifically around eliciting autoimmune responses. Disclosed herein are polypeptides, polynucleotides, compositions, and related methods that address this need.SUMMARY

[0006] In one aspect, disclosed herein are polynucleotides encoding a polypeptide, where the polypeptide comprises a viral endosomal sorting complex required for transport (ESCRT)- recruiting domain (ERD), where the viral ERD comprises a synthetic sequence derived from a virus. In some embodiments, the virus is selected from the group consisting of: equine infectious anemia virus (EIAV), human immunodeficiency virus (HIV), Ebola (EBOV), Mason-Pfizer monkey virus (MPMV), Human T-lymphotropic virus (HTLV), vaccinia virus (Vacc.), tick- borne encephalitis virus (TBEV), simian virus (SV-5), rous sarcoma virus, porcine endogenous retrovirus (PERV), prototype foamy virus (PFV), and feline immunodeficiency virus (FIV). In some embodiments, the sequence derived from the virus comprises an ESCRT-recruiting motif (ERM). In some embodiments, the ERM binds one or more ESCRT proteins. In certain embodiments, the one or more ESCRT proteins is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4-like protein.

[0007] In some embodiments of the polynucleotides encoding a polypeptide, where the polypeptide comprises a viral ERD, the ERM comprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL, LXXLF, PPXY, LXXLXXL, L(X)nLXXLXXL, PYXE, and YXXL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5. In some embodiments, the ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto.

[0008] In some embodiments of the polynucleotides encoding a polypeptide, where the polypeptide comprises a viral ERD, the viral ERD comprises the amino acid sequence of any one of SEQ ID NOs: 16-51 or an amino acid sequence having at least 70% sequence identity thereto.

[0009] In another aspect, disclosed herein are polynucleotides encoding a polypeptide, where the polypeptide comprises a tandem endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), wherein the tandem ERD comprises at least two ERDs. In some embodiments, the tandem ERD comprises a first ERD and a second ERD, where the first ERD comprises a first sequence derived from a first virus and the second ERD comprises a second sequence derived from a second virus. In some embodiments, the first and / or second virus is selected from the group consisting of: equine infectious anemia virus (EIAV), humanimmunodeficiency virus (HIV), Ebola (EBOV), Mason-Pfizer monkey virus (MPMV), Human T-lymphotropic virus (HTLV), vaccinia virus (Vacc.), tick-borne encephalitis virus (TBEV), simian virus (SV-5), rous sarcoma virus, porcine endogenous retrovirus (PERV), prototype foamy virus (PFV), and feline immunodeficiency virus (FIV). In certain embodiments, the first virus and the second virus are the same virus. In certain embodiments, the first virus and the second virus are not the same virus. In some embodiments, the first and / or second sequence comprises an ESCRT-recruiting motif (ERM). In certain embodiments, the ERM binds one or more ESCRT proteins. In certain embodiments, the one or more ESCRT proteins is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4-like protein. In certain embodiments, the ERM comprises a sequence selected from the group consisting of P(T / S)AP, YP(X)nL, LXXLF, PPXY, LXXLXXL, L(X)nLXXLXXL, PYXE, and YXXL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5.

[0010] In some embodiments, the ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto. In certain embodiments, the first sequence derived from a virus comprises a first ERM and the second sequence derived from a virus comprises a second ERM; where the first ERM is N-terminal to the second ERM; where the first ERM is PTAP, PSAP, PPXY, or YP(X)nL and the second ERM is LXXLF, LXXLXXL, LYPDLSEI, or IYPVRSNSTI; and where X is any amino acid and n is 1, 2, 3, 4, or 5. In certain embodiments, the first and / or the second ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto.

[0011] In some embodiments of the polynucleotides encoding a polypeptide where the polypeptide comprises a tandem ERD, the tandem ERD comprises a linker. In some embodiments, the linker is a flexible linker. In certain embodiments, the linker is a Glycine- Serine linker. In certain embodiments, the linker comprises the sequence of any one of SEQ ID NOs: 235-241.

[0012] In some embodiments of the polynucleotides encoding a polypeptide where the polypeptide comprises a tandem ERD, the tandem ERD comprises the amino acid sequence of any one of SEQ ID NOs: 52-116 or an amino acid sequence having at least 70% sequence identity thereto.

[0013] In another aspect, disclosed herein are polynucleotides encoding a polypeptide, where the polypeptide comprises a non-human homolog endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), where the non-human homolog ERD comprises atruncation relative to a naturally occurring non-human ERD. In some embodiments, the truncation relative to a naturally occurring non-human ERD comprises a truncation of 15 or less amino acids to the N terminus and / or the C terminus of a motif responsible for binding to one or more ESCRT proteins. In some embodiments, the naturally occurring non-human ERD is derived from a non-human vertebrate. In some embodiments, the non-human vertebrate is selected from the group consisting of: chicken, toad, mouse, elephant shrew, sunbittern, crocodile, Burmese python, fruit bat, pond turtle, and slow loris. In certain embodiments, the naturally occurring ERD is derived from a fish. In certain embodiments, the fish is selected from the group consisting of: zebrafish, greater pipefish, Atlantic halibut, snake pipefish, milkfish, European plaice, broad-nosed pipefish, banded pipefish, and goldfish.

[0014] In some embodiments of the polynucleotides encoding a polypeptide where the polypeptide comprises a non-human homolog ERD, the non-human homolog ERD comprises an amino acid sequence of SEQ ID NOs: 117-136, or an amino acid sequence having at least 70% sequence identity thereto, or a portion thereof.

[0015] In another aspect, disclosed herein are polynucleotides encoding a polypeptide, where the polypeptide comprises a synthetic endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), where the synthetic ERD comprises at least two ESCRT - recruiting motifs (ERMs). In some embodiments, the at least two ERMs bind to one or more proteins associated with ESCRT. In some embodiments, the one or more proteins associated with ESCRT is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4-like protein. In certain embodiments, each of the at least two ERM comprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL, LXXLF, PPXY, LXXLXXL, L(X)nLXXLXXL, PYXE, and YXXL, where X is any amino acid and wherein n is 1, 2, 3, 4, or 5. In certain embodiments, each ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto. In some embodiments of the polynucleotides encoding a polypeptide, where the polypeptide comprises a synthetic ERD, the synthetic ERD further comprises a linker. In some embodiments, the synthetic ERD comprises, from N-terminus to C-terminus:

[0016] a first ERM, a linker, and a second ERM;

[0017] a linker, a first ERM, a linker, a second ERM, and a linker;

[0018] a first ERM, a linker, a second ERM, a linker, and a third ERM;

[0019] a linker, a first ERM, a linker, a second ERM, a linker, a third ERM, and a linker;

[0020] a first ERM, a linker, a second ERM, a linker, a third ERM, a linker, and a fourth ERM; or

[0021] a linker, a first ERM, a linker, a second ERM, a linker, a third ERM, a linker, a fourth ERM, and a linker.

[0022] In some embodiments, the linker is a flexible linker. In certain embodiments, the linker is a Glycine-Serine (GS or SG) linker. In certain embodiments, the GS linker comprises the sequence of any one of SEQ ID NOs: 235-241. In some embodiments, the linker is a rigid linker. In certain embodiments, the rigid linker comprises the sequence of SEQ ID NO: 242 or SEQ ID NO: 243.

[0023] In some embodiments of the polynucleotides encoding a polypeptide, where the polypeptide comprises a synthetic ERD, the synthetic ERD comprises an ERM that binds to a NEDD4-like protein and an ERM that binds to ALIX, where the ERM that binds to a NEDD4- like protein is N-terminal to the ERM that binds to ALIX. In some embodiments, the synthetic ERD comprises a first ERM comprising a sequence of PTAP, PSAP, PPXY, or YP(X)nL and a second ERM comprising a motif different from that of the first ERM, where said first ERM is N-terminal to said second ERM. In some embodiments, the synthetic ERD comprises a first ERM comprising a sequence of LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI and a second ERM comprising a motif different from that of the first ERM, where said first ERM is C-terminal to said second ERM.

[0024] In some embodiments, the synthetic ERD comprises an ERM comprising a sequence of PTAP, PSAP, PPXY, or YP(X)nL and an ERM comprising a sequence of LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI, wherein said PTAP, PSAP, PPXY, or YP(X)nL sequence is N-terminal to said LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI sequence.

[0025] In some embodiments of the polynucleotides encoding a polypeptide where the polypeptide comprises a synthetic ERD, the synthetic ERD comprises the amino acid sequence of any one of SEQ ID NOs: 180-234 and 365-367, or an amino acid sequence having at least 70% sequence identity thereto.

[0026] In another aspect, disclosed herein are polynucleotides encoding a polypeptide, where the polypeptide comprises a synthetic coiled-coil endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), where the synthetic coiled-coil ERD comprises an amino acid sequence capable of binding one or more proteins associated with ESCRT; and where the synthetic coiled-coil ERD comprises a backbone capable of forming a coiled-coilstructure. In some embodiments, the one or more proteins associated with ESCRT is selected from the group consisting of: tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), and a NEDD4-like protein. In some embodiments, the one or more proteins associated with ESCRT is TSG101 and / or ALIX.

[0027] In some embodiments, the synthetic coiled-coil ERD is computationally designed based on the structure of the ESCRT- and ALIX-binding region (EABR) sequence from the human CEP55 protein. In some embodiments, the synthetic coiled-coil ERD has increased binding affinity to one or more proteins associated with ESCRT relative to the EABR sequence from the human CEP55 protein.

[0028] In some embodiments of the polynucleotides encoding a polypeptide, where the polypeptide comprises a synthetic coiled-coil ERD, the backbone comprises an amino acid sequence capable of forming an alpha helix. In some embodiments, the alpha helix interacts with another alpha helix formed by an identical sequence to make a 2-stranded coiled-coil structure. In some embodiments, the synthetic coiled-coil ERD comprises the amino acid sequence of any one of SEQ ID NOs: 140-179, 301-309, and 313-353, or an amino acid sequence having at least 70% sequence identity thereto.

[0029] In some embodiments of the polynucleotides encoding a polypeptide, where the polypeptide comprises a synthetic coiled-coil ERD, the backbone comprises a first animo acid sequence capable of forming an alpha helix and a second amino acid sequence capable of forming an alpha helix. In some embodiments, the first animo acid sequence capable of forming an alpha helix and the second amino acid sequence capable of forming an alpha helix are linked by a linker. In some embodiments, the linker links the C-terminus of the first animo acid sequence to the N-terminus of the second amino acid sequence. In certain embodiments, the linker is a flexible linker. In certain embodiments, the linker is a rigid linker. In some embodiments, the length of the linker is about 1 / 5 to 1 / 3 of the number of amino acids of the first or second amino acid sequence capable of forming an alpha helix. In some embodiments of the polynucleotides encoding a polypeptide, where the polypeptide comprises a synthetic coiled- coil ERD, the synthetic coiled-coil ERD comprises the amino acid sequence of any one of SEQ ID NOs: 244-250 and 349-353, or an amino acid sequence having at least 70% sequence identity thereto.

[0030] In some embodiments of the polynucleotides encoding a polypeptide, where the polypeptide comprises a synthetic coiled-coil ERD, the backbone comprises an amino acid sequence derived from yeast GCN4. In certain embodiments, the amino acid sequence derivedfrom yeast GCN4 comprises the sequence of any one of SEQ ID NOs: 137-139 or an amino acid sequence having at least 70% sequence identity thereto.

[0031] In some embodiments of the polynucleotides encoding a polypeptide, where the polypeptide comprises a synthetic coiled-coil ERD, the synthetic coiled-coil ERD comprises a chimeric sequence, wherein the chimeric sequence comprises a portion of a first ERD capable of forming a coiled-coil structure fused in frame to a portion of a second ERD capable of forming a coiled-coil structure. In certain embodiments, the first ERD and / or the second ERD is a nonhuman homolog ERD. In specific embodiments, the non-human homolog ERD comprises the sequence of any one of SEQ ID NOs: 119-136 or an amino acid sequence having at least 70% sequence identity thereto. In certain embodiments, the first ERD and / or the second ERD comprises the sequence of any one of SEQ ID NOs: 137-179, 252, 301-309, and 313-348, or an amino acid sequence having at least 70% sequence identity thereto.

[0032] The polynucleotide of claim 70, wherein the synthetic coiled-coil ERD comprises the amino acid sequence of SEQ ID NO: 348 or SEQ ID NO: 353 or an amino acid sequence having at least 70% sequence identity thereto.

[0033] In another aspect, disclosed herein are polynucleotides encoding a fusion protein, where the fusion protein comprises:(a) an antigenic polypeptide; and(b) an endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), where the ERD is:(i) a viral ERD;(ii) a tandem ERD;(iii) a non-human homolog ERD;(iv) a synthetic ERD comprising at least two ERMs; or(v) a synthetic coiled-coil ERD.

[0034] In some embodiments, the antigenic polypeptide is derived from a protein of a virus. In some embodiments, the virus is hMPV, PIV3, respiratory syncytial virus, VZV, CMV, HSV1, HSV2, EBV, a coronavirus, influenza, a flavivirus, or orthopoxvirus. In some embodiments, the coronavirus is MERS-CoV, SARS-CoV, or SARS-CoV-2. In certain embodiments, the protein is spike protein and the virus is MERS-CoV. In certain embodiments, the protein is spike protein and the virus is SARS-CoV. In certain embodiments, the protein is spike protein and the virus is SARS-CoV-2. In certain embodiments, the protein is fusion (F) protein and the virus is hMPV. In certain embodiments, the protein is fusion (F) protein and thevirus is PIV3. In certain embodiments, the protein is fusion (F) protein and the virus is respiratory syncytial virus. In certain embodiments, the protein is glycoprotein E (gE) and the virus is VZV. In certain embodiments, the protein is glycoprotein H (gH), glycoprotein L (gL), or glycoprotein B (gB) and the virus is CMV. In certain embodiments, the protein is glycoprotein C (gC) or glycoprotein D (gD) and the virus is HSV1 or HSV2.

[0035] In some embodiments of the polynucleotides encoding a fusion protein, the fusion protein comprises an antigenic polypeptide derived from a protein of a bacterium. In some embodiments, the bacterium is an acne-causing bacterium, Staphylococcus, Borrelia, E. Coli, or Chlamydia. In certain embodiments, the protein is DsAl and the acne-causing bacterium is Cutibacterium acnes. In certain embodiments, the protein is esxA or esxB and the bacterium is Staphylococcus. In certain embodiments, the protein is OspA and the bacterium is Borrelia, optionally Borrelia burgdorferi . In certain embodiments, the protein is FimH and the bacterium is E. Coli. In certain embodiments, the protein is major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), or OmcB and the bacterium is Chlamydia.

[0036] In some embodiments of the polynucleotides encoding a fusion protein, the fusion protein comprises a viral ERD comprising a sequence derived from a virus. In some embodiments, the fusion protein comprises a tandem ERD comprising at least two ERDs. In some embodiments, the fusion protein comprises a non-human homolog ERD. In some embodiments, the fusion protein comprises a synthetic ERD comprising at least two ESCRT- recruiting motifs (ERMs). In some embodiments, the fusion protein comprises a synthetic coiled-coil ERD, where the synthetic coiled-coil ERD comprises an amino acid sequence capable of binding one or more proteins associated with ESCRT, where the synthetic ERD comprises a backbone capable of forming a coiled-coil structure.

[0037] In some embodiments of the polynucleotides of the disclosure, the polynucleotide comprises RNA. In certain embodiments, the polynucleotide comprises mRNA. In some embodiments, the polynucleotide comprises DNA.

[0038] In another aspect, disclosed herein are polypeptides encoded by a polynucleotide of the disclosure.

[0039] In yet another aspect, disclosed herein is a cell expressing on its surface the fusion protein, or a portion of the fusion protein, encoded by a polynucleotide of the disclosure.

[0040] In another aspect, disclosed herein is an enveloped virus-like particle (eVLP) comprising the fusion protein encoded by a polynucleotide of the disclosure. Also disclosed herein is an eVLP displaying on its surface the antigenic polypeptide, or part of the antigenicpolypeptide, of the fusion protein encoded by a polynucleotide of the disclosure. In some embodiments of the eVLP of the disclosure, the diameter of the eVLP is about 10 nm - about 150 nm.

[0041] In another aspect, disclosed herein are vectors comprising a polynucleotide of the disclosure. In some embodiments, the vector is a viral vector. 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). In certain embodiments, the non-viral vector is a lipid nanoparticle (LNP) and the polynucleotide comprises mRNA.

[0042] In another aspect, disclosed herein is a method of preventing or treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a polynucleotide of the disclosure, an eVLP of the disclosure, a polypeptide of the disclosure, or a vector of the disclosure. In certain embodiments, the subject is a human subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1. depicts a schematic of cells producing eVLPs in ESCRT-dependent fashion in which a fraction of the protein is presented on the cell surface and a different fraction is presented on the eVLPs.

[0044] FIG. 2A depicts a schematic depicting an antigenic peptide (e.g., an exemplary MERS spike protein) with an ERD at the C terminus as either a linear schematic (left) or a cartoon of the C-terminus including the transmembrane domain (right) showing the location of ERD attachment.

[0045] FIG. 2B depicts a linear schematic depicting the expression of viral ERDs derived from viral Late Domains, gag proteins, etc. at the C terminus of MERS spike protein.

[0046] FIG. 2C depicts a linear schematic depicting the expression of tandem viral ERDs derived from viral Late Domains, gag proteins, etc. at the C terminus of MERS spike protein.

[0047] FIG. 2D depicts a linear schematic depicting the expression of homologous EABR sequences from the CEP55 protein of other vertebrates at the C terminus of MERS spike protein.

[0048] FIG. 2E depicts a linear schematic depicting the expression of synthetic ERD sequences derived from grafting residues of the EABR protein onto the GCN4 protein derived from yeast.

[0049] FIG. 3 depicts a schematic of the synthetic ERD sequences generated as a chimera by grafting residues of the EABR protein onto the GCN4 protein.

[0050] FIG. 4A depicts a schematic of a plate layout containing ERD sequences of the disclosure designed as disclosed herein. The ERD sequences were positioned at the C-terminus of a truncated MERS spike protein which contains a transmembrane domain and a short cytoplasmic domain (as depicted in FIGS. 2A-2E).

[0051] FIG. 4B depicts a dot blot measuring supernatant of HEK293T cells transfected with the ERD sequences as detailed in aspects of this disclosure. The order of the transfected sequences is as in FIG 3.

[0052] FIG. 5 depicts a replicate of FIG. 4B but conducted in a mouse 3T3 cells.

[0053] FIG. 6 depicts a plot reflecting the dot blot density on the y-axis (derived fromFIG. 4B and its replicates) to the total cell expression, as determined by a cell ELISA of fixed and permeabilized cells, on the x-axis. Quadrants representing high / low ERD activity (dot blot density correlating to budded eVLPs) and high / low expression (ELISA signal) are denoted. Symbol color is separated by ERD type (as outlined in FIGS. 2A-2E).

[0054] FIG. 7 depicts a subset of the plot in FIG. 6 that was selected for being a diverse set of ERDs from different quadrants,

[0055] FIG. 8 depicts a graph of dynamic light scattering, a tool to measure size of particles, was employed on purified eVLPs, a subset of which are shown in FIG 7.

[0056] FIG. 9A depicts a graph of dynamic light scattering, as in FIG. 8, but specific for WT human CEP55 EABR. The dotted line is the peak radius for the WT CEP55 EABR.

[0057] FIG. 9B depicts a graph of dynamic light scattering, as in FIG. 8, but specific for a truncated form of human CEP55 EABR. The dotted line is the peak radius for the WT CEP55 EABR.

[0058] FIG. 9C depicts a graph of dynamic light scattering, as in FIG. 8, but specific for a tandem viral ERD from MPMV and El AV. The dotted line is the peak radius for the WT CEP55 EABR.

[0059] FIG. 9D depicts a graph of dynamic light scattering, as in FIG. 8, but specific for a tandem viral ERD from EBOV and HIV. The dotted line is the peak radius for the WT CEP55 EABR

[0060] FIG. 9E depicts a graph of dynamic light scattering, as in FIG. 8, but specific for a tandem viral ERD from MPMV and HIV. The dotted line is the peak radius for the WT CEP55 EABR.

[0061] FIG. 9F depicts a graph of dynamic light scattering, as in FIG. 8, but specific for a tandem viral ERD from Vaccina virus and EBOV. The dotted line is the peak radius for the WT CEP55 EABR.

[0062] FIG. 9G depicts a graph of dynamic light scattering, as in FIG. 8, but specific for a tandem viral ERD from HIV and SV. The dotted line is the peak radius for the WT CEP55 EABR.

[0063] FIG. 9H depicts a graph of dynamic light scattering, as in FIG. 8, but specific for truncated Pond Turtle EABR. The dotted line is the peak radius for the WT CEP55 EABR.

[0064] FIG. 91 depicts a graph of dynamic light scattering, as in FIG. 8, but specific for truncated Slow Loris EABR. The dotted line is the peak radius for the WT CEP55 EABR

[0065] FIG. 9J depicts a graph of dynamic light scattering, as in FIG. 8, but specific for truncated Fruit Bat EABR. The dotted line is the peak radius for the WT CEP55 EABR.

[0066] FIG. 9K depicts a graph of dynamic light scattering, as in FIG. 8, but specific for a synthetic ERD based on a chimera of GCN4 and human CEP55 EABR. The dotted line is the peak radius for the WT CEP55 EABR.

[0067] FIG. 10 depicts a SDS-Page gel of isolated eVLPs, as selected from FIG. 7 and purified via StrepTactin® as above, were run on a reducing SDS-PAGE gel. Similar rank order of purified proteins was determined from this analysis as expected from FIG. 4B and FIG. 7.

[0068] FIG. 11 depicts a transmission electron cryomicroscopy (CryoTEM) images of purified eVLPs containg the MERS spike protein with human CEP55 EABR show fairly homogenous coronas of spike protein on eVLPs of anticipated sizes as per the DLS data

[0069] FIG. 12A depicts a dot blot analysis, similar to that conducted in FIG. 4B, but with a narrowed set of ERDs (some of which outlined in FIG. 7) against a different antigen, gB from EBV. gB, like MERS spike, is a trimer and shows a similar rank order of ERDs compared to the MERS spike protein.

[0070] FIG. 12B depicts a dot blot analysis, similar to that conducted in FIG. 4B, but with a narrowed set of ERDs (some of which outlined in FIG. 7) against a different antigen, gp220 from EBV. Gp220, unlike the MERS spike protein and gB, is a monomer and shows a unique rank order of the ERDs compared to the MERS spike protein.

[0071] FIG. 13 depicts a graph of ERD activity, as measured by mean dot blot intensity, of various viral tandem ERDs or non-tandem ERDs in the middle of each plot labeled “solo”.

[0072] FIG. 14 depicts a graph of eVLP inducing activity, as measured by mean dot blot intensity, of engineered viral tandem ERDs and synthetic ERDs. The x axis depicts the total protein expression in the cells by cell ELISA.

[0073] FIG. 15 depicts a schematic of the design of synthetic ERDs and the motifs used for generating the ERDs.

[0074] FIG. 16 depicts a graph of eVLP inducing activity, as measured by mean dot blot intensity, of sequences generated through ERD-informed designs compared to that of sequences generated through random designs. Also shown is a graph depicting the total protein expression as measured by cell ELISA.

[0075] FIG. 17 depicts a dot blot analysis against hMPV F protein in supernatant collected from Expi 293F transfected with various constructs, each containing a different ERD sequence.

[0076] FIG. 18 depicts a graph showing the quantitation, through densitometry analysis, of the dot blot against hMPV F protein of FIG. 17.

[0077] FIG. 19 depicts immunization data from ERD-tagged hMPV F antigens after animals received a single prime dose (left) or a prime dose and a boost dose (right) of various constructs, with two containing a different ERD sequence and a cell anchored hMPV F antigenic polypeptide.

[0078] FIG. 20 depicts a dot blot analysis against VZV gE protein in supernatant collected from Expi 293F transfected with various constructs, each containing a different ERD sequence.

[0079] FIG. 21 depicts a graph showing the quantitation, through densitometry analysis, of the dot blot against VZV gE protein of FIG. 20.

[0080] FIG. 22 depicts immunization data from ERD-tagged VZV gE antigens after animals received a single prime dose (left) or a prime dose and a boost dose (right) of various constructs, each containing a different ERD sequence and a cell anchored VZV gE antigenic polypeptide.

[0081] FIG. 23 depicts scatterplots and histogram showing a gating strategy for the analysis of the distribution of ERD-expressing cells via flow cytometry.

[0082] FIG. 24 depicts scatterplots showing the analysis of the distribution of ERD- expressing cells in mock transfected cells (left) and cells transfected with human EABR (right) via flow cytometry.DETAILED DESCRIPTIONTerms and Concepts

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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. 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. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated. When "about" is applied to the beginning of a numerical range, it applies to both ends of the range.

[0087] The terms "protein" and "polypeptide" are used interchangeably 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 terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0088] The terms "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 sequence 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 sequencesimilarity 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.

[0089] 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.

[0090] % Query sequence identity: = (Number of alignment identities) / (Length of Query sequence);

[0091] % Subject sequence identity: = (Number of alignment identities) / (Length ofSubject sequence);

[0092] %Alignment sequence identity = (Number of alignment identities) / (Length of Alignment)

[0093] Accordingly, when the term “sequence identity” is used herein, it can include any of the above non-limiting methodologies provided above to calculate.

[0094] The term "antigenic" and the related term, such as “immunogenic”, when used in the context of the present disclosure, refers to the ability of an antigen, which can be a protein, a polypeptide, or a region of a protein or a polypeptide, or a nucleic acid encoding a polypeptide, to elicit in a subject an immune response to the specific antigen. Various aspects of an immune response elicited by antigenic compositions can be determined using standard assays, some of which are described in the present disclosure.

[0095] Disclosed herein are ESCRT-dependent methods of producing enveloped particles in the context of delivery of genetic material in a vaccine setting encoded, for example, by DNA or RNA. Specifically, polynucleotides that encode the fusion proteins that comprise antigenic polypeptides and ESCRT recruiting domains (ERDs) are described. The polynucleotides of the disclosure encoding fusion proteins that comprise antigenic polypeptides and ERDs capable of being expressed on enveloped particles and corresponding features described throughout the application. Numerous embodiments of non-human ERDs are described.Overview

[0096] Provided herein are a series of innovative ESCRT recruiting domains (ERDs) that when expressed with an antigenic polypeptide, may be useful as a vaccine to treat or prevent a disease or disorder. Also provided are fusion proteins comprising an antigenic polypeptide and an ERD disclosed herein. Without being bound to theory or mechanism, in some embodiments, the ERDs facilitate ESCRT protein recruitment and allow for spontaneous production ofenveloped particles referred to herein as enveloped virus-like particles (eVLP) and the antigenic polypeptides (or part of the antigenic polypeptides) are displayed on the surface of the eVLPs.

[0097] Additionally, provided herein are polynucleotides encoding the ERDs described herein and polynucleotides encoding the fusion proteins described herein. In some embodiments, the polynucleotides comprises DNA or RNA (including mRNA). In some embodiments, the polynucleotides of the disclosure may be administered to a subject as a vaccine composition to prevent a disease or disorder. Also provided herein are polypeptides encoded by any one of the polynucleotides described herein.

[0098] Furthermore, provided herein are vectors comprising the polynucleotides disclosed herein and cells and eVLPs comprising the ERDs, fusion proteins, or polynucleotides described herein.I. Non-Human ESCRT-recruiting domains

[0099] In one aspect, provided herein are several categories of non-human ERDs: (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. These categories of ERDs are described in further detail below.

[0100] In some embodiments, a viral ERD described herein comprises a sequence derived from a virus.

[0101] In some embodiments, a non-human homolog ERD described herein comprises a truncation relative to a naturally occurring non-human ERD.

[0102] In some embodiments, a synthetic coiled-coil ERD described herein comprises a backbone capable of forming a coiled-coil structure and comprises an amino acid sequence capable of binding one or more proteins associated with ESCRT.

[0103] In some embodiments, a synthetic ERD with ERMs described herein comprises at least two ERMs.

[0104] In some embodiments, a tandem ERD described herein comprises a plurality of any of the ERDs described herein. In some embodiments, the tandem ERD comprises a first ERD and a second ERD. In certain embodiments, the first ERD is a first viral ERD and the second ERD is a second viral ERD. A tandem ERD comprising a first and a second viral ERD is also herein referred to as a viral tandem ERD.A, Viral ERD

[0105] In one aspect, disclosed herein are viral ERDs comprising a sequence derived from a virus. In some embodiments, the viral ERDs described herein comprise a sequence derived from a virus selected from the group consisting of: equine infectious anemia virus (EIAV), human immunodeficiency virus (HIV), Ebola (EBOV), Mason-Pfizer monkey virus (MPMV), Human T-lymphotropic virus (HTLV), vaccinia virus (Vacc), tick-borne encephalitis virus (TBEV), simian virus (SV-5), rous sarcoma virus, respiratory syncytial virus, porcine endogenous retrovirus (PERV), mouse mammary tumor virus (MMTV), prototype foamy virus (PFV), feline immunodeficiency virus (FIV), lymphocytic choriomeningitis virus (LCMV), PEAV (porcine enteric alphacoronavirus), simian immunodeficiency virus (SIV), human metapneumovirus (hMPV), human T Cell Leukemia Virus, Bovine Foamy Virus, mumps virus, vesicular stomatitis virus (VSV), and Mammarenavirus lassaense.

[0106] In some embodiments, the sequence derived from the virus comprises an ESCRT - recruiting motif (ERM). An ERM is an amino acid motif capable of recruiting a cell’s ESCRT machinery, thereby facilitating an ERD’s capability to induce ESCRT-mediated budding of eVLPs. In some embodiments, the ERM binds, engages, or recruits one or more ESCRT proteins (e.g. TSG101, ALIX, and / or NEDD4). In some embodiments, the one or more ESCRT proteins is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4-like protein. In certain embodiments, the one or more ESCRT proteins is TSG101. In certain embodiments, the one or more ESCRT proteins is ALIX. In certain embodiments, the one or more ESCRT proteins is a NEDD4-like protein. A NEDD4-like protein may refer to one of nine currently known members in the human NEDD4 family, including NEDD4, NEDD4-2 (NEDD4L), ITCH, SMURF1, SMURF2, WWP1, WWP2, NEDL1, and NEDL2. It can be appreciated that the one or more ESCRT proteins may also be a protein in the human NEDD4 family that has yet to be identified.

[0107] In some embodiments, a viral ERD described herein comprises at least one, at least two, at least three, or at least four ESCRT -recruiting motifs (ERMs). In some embodiments, the viral ERD comprises at least two ERMs. In some embodiments, the viral ERD comprises at least three ERMs. In some embodiments, the viral ERD comprises at least four ERMs. In some embodiments, the viral ERD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ESCRT-recruiting motifs (ERMs). In certain embodiments, the viral ERD comprises 1 ERM. In certain embodiments, the viral ERD comprises 2 ERMs. In certain embodiments, the viral ERD comprises 3 ERMs. In certain embodiments, the viral ERD comprises 4 ERMs. In some embodiments, the ERMcomprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL, LXXLF, PPXY, L(X)nLXXLXXL, FPIV, PYXE, YXXL, LYPDLSEI, PLPPV, YEIL, and PYKELYPL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5.

[0108] In some embodiments, the ERM comprises a sequence in Table 2B. In some embodiments, the ERM comprises a sequence in Table 2A. In certain embodiments, the ERM is derived from a Late Domain. A Late Domain is a portion of a virus, for example the gag protein in a retrovirus, that is responsible for budding off the cell and acting late in viral replication. Late domains often contain ERMs that are responsible for engaging host cell machinery to facilitate ESCRT-mediated viral budding. Without being held to theory or mechanism, in some embodiments, ERDs that comprise an ERM and / or a Late Domain may engage ESCRT machinery to a lesser extent than human ERDs or viral ERDs without an ERM or a Late Domain, which may allow the non-human ERDs with an ERM and / or a Late Domain to avoid rapid budding prior to proper eVLP formation. In some embodiments, the viral ERDs disclosed herein comprises an ERM derived from a Late Domain. In some embodiments, the viral ERDs disclosed herein comprise a Late Domain. In some embodiments, the Late Domain binds, engages, or recruits one or more ESCRT proteins (e.g. TSG101, ALIX, and / or NEDD4). In some embodiments, the one or more ESCRT proteins is TSG101, ALIX, or a NEDD4-like protein. In certain embodiments, the one or more ESCRT proteins is TSG101. In certain embodiments, the one or more ESCRT proteins is ALIX. In certain embodiments, the one or more ESCRT proteins is a NEDD4-like protein.

[0109] Exemplary ESCRT-recruiting motifs are provided in Table 2A and Table 2B below. In some embodiments, the ERM comprises any one of the sequences in Table 2A or Table 2B, or a sequence with at least 70% sequence identity thereto. In some embodiments, the ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0110] In some embodiments, the viral ERD described herein comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92%sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.[OHl] In some embodiments, the viral ERD described herein comprises the amino acid sequence of any one of SEQ ID NOs: 16-51 or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0112] Table 2A provides sequences for exemplary ESCRT-recruiting motifs (ERMs) that are derived from viral Late Domains. These ERMs may be used in any of the ERDs described herein. It can be appreciated that the amino acid residues which mediate binding to one or more ESCRT proteins may be identified from the exemplary ERMs presented in Table 2A. The inclusion of such amino acid residues in an ERM described herein may be sufficient to preserve the function of an ERD comprising said ERM.Table 2 A: Amino acid sequences of exemplary ESCRT-recruiting motifs derived from viral LateDomains

[0113] Table 2B provides sequences for exemplary ESCRT-recruiting motifs that may be used in any of the ERDs described herein.Table 2B: Amino acid sequences of exemplary ESCRT recruiting motifs

[0114] Table 2C provides sequences for exemplary ERDs that comprise an ESCRT- recruiting motif. These ERDs may be used in any of the polypeptides or fusion proteins described hereinTable 2C: Exemplary ERDs that comprise an ESCRT-recruiting motif

[0115] Also disclosed herein are polypeptides comprising a viral ERD described herein. It can be appreciated that any viral ERD that comprises an ERM or a Late Domain described herein may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, which is further discussed below in detail. Provided herein are polypeptides comprising a sequence in Table 2A, Table 2B, or Table 2C.

[0116] Additionally, disclosed herein are polynucleotides encoding the polypeptides comprising a viral ERD and polynucleotides encoding the fusion proteins comprising a viral ERD. In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprises an antigenic polypeptide described herein and a viral ERD. Provided herein are polynucleotides that encode any one or more of the sequences of Table 2A, Table 2B, and Table 2C, or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0117] . Polynucleotides can be DNA or RNA (e.g., mRNA), as will be described in more detail herein.B, Non-Human Homolog ERDs

[0118] In another aspect, disclosed herein are non-human homolog ERDs comprising a truncation relative to a naturally occurring non-human ERD. A truncation relative to a naturallyoccurring non-human ERD may comprise truncation of 15 or less amino acids to the N terminus and / or the C terminus of a motif responsible for binding to ALIX and TSG101, such as an ESCRT-recruiting motif (ERM). In some embodiments, the truncation comprises deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids to the N terminus and / or the C terminus of a motif responsible for binding to ALIX and TSG101, such as an ESCRT-recruiting motif (ERM).

[0119] An ESCRT- and ALIX-binding region (EABR) sequence may be used to recruit ESCRT proteins to induce the budding of eVLP. In some embodiments, a non-human homolog ERD described herein comprises an EABR sequence. The residues in EABR that engage ESCRT proteins (e.g. TSG101 and / or ALIX) may comprise amino acid sequences derived from one or more non-human vertebrate ERDs. In certain embodiments, the EABR sequence is derived from a non-human vertebrate. In certain embodiments, the EABR sequence is derived from a fish. In some embodiments, a non-human homolog ERD described herein is derived from a non-human vertebrate. In certain embodiments, the vertebrate is selected from the group consisting of: chicken, toad, mouse, elephant shrew, sunbittem, crocodile, Burmese python, fruit bat, pond turtle, and slow loris. In some embodiments, a non-human homolog ERD described herein is derived from a fish. In certain embodiments, the fish is selected from the group consisting of: zebrafish, greater pipefish, Atlantic halibut, snake pipefish, milkfish, European plaice, broad-nosed pipefish, banded pipefish, and goldfish.

[0120] In some embodiments, the non-human homolog ERDs described herein comprises a truncation relative to a naturally occurring non-human ERD, wherein the truncation generates an amino acid sequence of similar length as that of an EABR sequence. In some embodiments, the truncation generates an amino acid sequence that remains capable of forming an alpha helix. The naturally occurring non-human ERD may be derived from a non-human vertebrate or a fish. In some embodiments, the naturally occurring non-human ERD is derived from a vertebrate. In certain embodiments, the vertebrate is selected from the group consisting of: chicken, toad, mouse, elephant shrew, sunbittern, crocodile, Burmese python, fruit bat, pond turtle, and slow loris. In some embodiments, the naturally occurring non-human ERD is derived from a fish. In certain embodiments, the fish is selected from the group consisting of: zebrafish, greater pipefish, Atlantic halibut, snake pipefish, milkfish, European plaice, broad-nosed pipefish, banded pipefish, and goldfish.

[0121] In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring chicken ERD sequence. In some embodiments, the non-humanhomolog ERD comprises a truncation relative to a naturally occurring toad ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring mouse ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring elephant shrew ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring sunbittern ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring crocodile ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring Burmese python ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring fruit bat ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring pond turtle ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring slow loris ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring zebrafish ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring greater pipefish ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring Atlantic halibut ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring snake pipefish ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring milkfish ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring European plaice ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring goldfish ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring broad-nosed pipefish ERD sequence. In some embodiments, the non-human homolog ERD comprises a truncation relative to a naturally occurring banded pipefish ERD sequence.

[0122] In some embodiments, the non-human homolog ERD described herein comprises the amino acid sequence of any one of SEQ ID NOs: 119-136, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0123] In some embodiments, the non-human homolog ERD described herein comprises any one of the amino acid sequences in Table 3A or Table 3B, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0124] Table 3A provides exemplary amino acid sequences for modified non-human vertebrate ERDs.Table 3A: Exemplary non-human vertebrate ERD amino acid sequences

[0125] Table 3B provides exemplary amino acid sequences for modified fish ERDs.Table 3B: Exemplary fish ERD amino acid sequences

[0126] Also disclosed herein are polypeptides comprising a non-human homolog ERD described herein. It can be appreciated that the non-human homolog ERDs described herein may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, further discussed below in detail. Provided herein are polypeptides comprising a sequence in Table 3A or Table 3B or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0127] Additionally, disclosed herein are polynucleotides encoding the polypeptides comprising a non-human homolog ERD, and disclosed herein are polynucleotides encoding the fusion proteins comprising a non-human homolog ERD. In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprisesan antigenic polypeptide described herein and a non-human homolog ERD. Provided herein are polynucleotides that encode any one or more of the sequences of Table 3A and Table 3B, or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.. Polynucleotides can be DNA or RNA (e.g., mRNA), as will be described in more detail herein.C. Synthetic coiled-coil ERDs

[0128] An ERD may utilize a particular structure that facilitates the binding, engagement, or recruitment of the one or more ESCRT proteins (e.g. TSG101, ALIX, and / or NEDD4). For example, an ERD may adopt an alpha helix structure or a coiled-coiled structure. Disclosed herein are synthetic coiled-coil ERDs that are generated to comprise a backbone amino acid sequence capable of forming a coiled-coil structure, wherein the backbone sequence may optionally be modified to comprise a plurality of amino acid residues that facilitate the binding of one or more ESCRT proteins (e.g. TSG101, ALIX, and / or NEDD4). In some embodiments, the synthetic coiled-coil ERD comprises an amino acid sequence capable of binding one or more proteins associated with ESCRT. In some embodiments, the synthetic coiled-coil ERDs disclosed herein comprise a backbone amino acid sequence that is capable of forming a coiled- coil structure. In some embodiments, the amino acid sequence capable of binding one or more proteins associated with ESCRT is part of the backbone capable of forming a coiled-coil structure. In some embodiments, the backbone is modified to comprise a plurality of amino acid residues that facilitate the binding of one or more ESCRT proteins.

[0129] A coiled-coil structure is generally composed of two or more alpha helices. Without being held to theory or mechanism, in some embodiments, the synthetic ERDs disclosed herein are capable of forming a coiled-coil structure because they comprise at least one sequence that is capable of forming an alpha helix. Two copies of a synthetic coiled-coil ERD disclosed herein, or two copies of a polypeptide comprising a synthetic coiled-coil ERD, may each provide one alpha helix for the formation of the coiled-coil structure. Without being held to theory or mechanism, in some embodiments, assembly of the alpha helices into a coiled- coil structure presents a binding interface for proteins associated with ESCRT (e.g. TSG101, ALIX, and / or NEDD4). In some embodiments, the sequence capable of forming a coiled-coilstructure is derived from a naturally occurring amino acid sequence that is capable of forming a coiled-coil structure. The naturally occurring amino acid sequence may be isolated or derived from the yeast transcriptional activator GCN4, or homologs thereof. In some embodiments, the sequence capable of forming a coiled-coil structure comprises a fragment of or an amino acid sequence isolated or derived from the amino acid sequence of GCN4: MSEYQPSLFALNPMGFSPLDGSKSTNENVSASTSTAKPMVGQLIFDKFIKTEEDPIIKQDT PSNLDFDFALPQTATAPDAKTVLPIPELDDAVVESFFSSSTDSTPMFEYENLEDNSKEWT SLFDNDIPVTTDDVSLADKAIESTEEVSLVPSNLEVSTTSFLPTPVLEDAKLTQTRKVKKP NSVVKKSHHVGKDDESRLDHLGVVAYNRKQRSIPLSPIVPESSDPAALKRARNTEAARR SRARKLQRMKQLEDKVEELLSKNYHLENEVARLKKLVGER (SEQ ID NO: 251). In some embodiments, the synthetic ERDs described herein comprise a fragment of or an amino acid sequence isolated or derived from the sequence of SEQ ID NO: 251.

[0130] In some embodiments, a plurality of amino acid residues capable of binding one or more ESCRT proteins (e.g. TSG101, ALIX, and / or NEDD4) may be incorporated into the backbone capable of forming a coiled-coil structure, as depicted in FIG. 3. In some embodiments, the plurality of amino acid residues incorporated into the backbone may be about 1% to about 80% of the total amino acid residues within the synthetic ERD. In some embodiments, the plurality of amino acid residues incorporated into the backbone coiled-coil structure may be about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 30%, about 1% to about 35%, about 1% to about 40%, about 1% to about 45%, about 1% to about 50%, about 1% to about 55%, about 1% to about 60%, about 1% to about 65%, about 1% to about 70%, about 1% to about 75%, or about 1% to about 80% of the total amino acid residues within the synthetic ERD. In some embodiments, the plurality of amino acid residues incorporated into the backbone coiled-coil structure is about 1%, about 2%, about 3%, about 4 %, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total amino acid residues within the synthetic ERD.

[0131] Additionally, synthetic coiled-coil ERDs described herein may be designed using deep learning-based protein sequence design methods, such as Protein MPNN (PMPNN). In some embodiments, these computationally generated synthetic coiled-coil ERDs, like the ERDs comprising a sequence isolated or derived from GCN4, are designed to retain the presumed coiled-coil structure of the human EABR sequence, while retaining minimal to no humansequences. In some embodiments, computational tools are utilized to redevelop the coiled-coil structure and retain amino acid residues that facilitate the coiled-coil’ s interaction with host ESCRT machinery, such as TSG101, ALIX, and / or NEDD4. In some embodiments, the synthetic coiled-coil ERDs described herein are designed based on the structure of the EABR sequence from a CEP55 protein. In certain embodiments, the CEP55 protein is human CEP55. In some embodiments, the synthetic coiled-coil ERDs described herein are computationally predicted to fold like the human EABR sequence from CEP55. In some embodiments, synthetic coiled-coil ERD sequences that are generated using sequence design methods are computationally predicted to have a backbone capable of forming a coiled-coil structure. For example, a synthetic coiled-coil ERD described herein may comprise a sequence capable of forming an alpha helix which interacts with another alpha helix formed by a sequence of a naturally occurring ERD or a sequence from another synthetic coiled-coil ERD, thereby forming a coiled-coil structure.

[0132] Additionally, a synthetic coiled-coil ERD described herein may be capable of forming a coiled-coil structure because it comprises at least one sequence that is capable of forming an alpha helix. Two copies of a synthetic ERD disclosed herein may each provide one alpha helix for the formation of a coiled-coil structure. Alternatively, a synthetic coiled-coil ERD described herein may comprise two sequences, each capable of forming an alpha helix, and the two alpha helices interact to form a coiled-coil structure. Without being held to theory or mechanism, in some embodiments, assembly of the alpha helices into a coiled-coil structure presents a binding interface for proteins associated with ESCRT, thereby creating a coiled-coil structure capable of recruiting, binding, or engaging one ore more proteins associated with ESCRT (e g. TSG101, ALIX, and / or NEDD4).

[0133] In some embodiments, the synthetic coiled-coil ERD described herein comprises a backbone comprising a sequence capable of forming an alpha helix. In some embodiments, the synthetic coiled-coil ERD described herein comprises a backbone comprising a first sequence capable of forming an alpha helix and a second sequence capable of forming an alpha helix. In certain embodiments, the first and the second sequence capable of forming an alpha helix are capable of interacting with each other to form a coiled-coil structure. In some embodiments, the first sequence capable of forming an alpha helix and the second sequence capable of forming an alpha helix are the same. In some embodiments, the first sequence capable of forming an alpha helix and the second sequence capable of forming an alpha helix are different. In some embodiments, the two alpha helices formed by the first and the second sequence interact to forma coiled-coil structure. In certain embodiments, the two alpha helices form a parallel coiled-coil. In certain embodiments, the two alpha helices form an antiparallel coiled-coil.

[0134] In some embodiments, the synthetic coiled-coil ERDs described herein comprise a linker and a backbone comprising a first sequence capable of forming an alpha helix and a second sequence capable of forming an alpha helix. In some embodiments, the first sequence capable of forming an alpha helix and the second sequence capable of forming an alpha helix are linked by a linker. In some embodiments, the linker links the C-terminus of the first sequence to the N-terminus of the second sequence. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker. In some embodiments, the length of the linker is about 1 / 5 to 1 / 3 of the number of amino acids of the first or second amino acid sequence capable of forming an alpha helix. In certain embodiments, the length of the linker is about 1 / 5 of the number of amino acids of the first or second amino acid sequence capable of forming an alpha helix. In certain embodiments, the length of the linker is about 1 / 4 of the number of amino acids of the first or second amino acid sequence capable of forming an alpha helix. In certain embodiments, the length of the linker is about 1 / 3 of the number of amino acids of the first or second amino acid sequence capable of forming an alpha helix. Without being held to theory or mechanism, in some embodiments, the linker is long enough to connect the C-terminus of one alpha helix (coil) to the N-terminus of the other alpha helix (coil) while still allowing the formation of the coiled-coil structure. In some embodiments, the linker is flanked by an amino acid (e.g. proline) at either end to facilitate breaking of the alpha helical structure of the first and / or the second sequence capable of forming an alpha helix. In some embodiments, the linker is flanked by a single proline at both ends to facilitate breaking of the alpha helical structure of the first and / or the second sequence capable of forming an alpha helix.

[0135] The first sequence and / or the second sequence capable of forming an alpha helix may be truncated, thereby allowing for the use of a shorter linker to connect the sequences while still allowing for the formation of two alpha helices and a coiled-coil structure. The synthetic coiled-coil ERDs described herein may be engineered to improve its ability to recruit, bind, or engage a protein associated with ESCRT, for example, by increasing its affinity for an ESCRT associated protein. As an example, amino acid residues or sequence motifs that facilitate the binding of the synthetic coiled-coil ERD to an ESCRT protein may be substituted or inserted into the synthetic ERD sequence. In some embodiments, an ERM is inserted into a synthetic coiled-coil ERD described herein. In some embodiments, a sequence in a synthetic coiled-coil ERD described herein is substituted for an ERM.

[0136] Without being held to theory or mechanism, in some embodiments, human EABR forms a dimer which facilitates its function in the ESCRT pathway (e.g. recruit ESCRT proteins). The dimerization of EABR is generally spontaneous . However, in some embodiments, such spontaneous dimerization may not occur when an EABR is fused to a foreign protein, such as an antigen described herein. Accordingly, and in order to address this, in some embodiments single-chain ERD sequences comprising two copies of an ERD sequence are created, optionally by introducing a linker to link the two copies. In certain embodiments, single-chain ERD sequences comprising two copies of an EABR sequence are created, optionally by introducing a linker to link the two copies. A single chain sequence with more than one ERD copy may facilitate dimerization of ERDs and recruitment of ESCRT proteins. For example, described herein are single-chain ERD sequences comprising two copies of an EABR or two copies of a short EABR sequence, referred to as EABR SC (SEQ ID NO: 349) and EABR SC short (SEQ ID NO: 350), respectively, that were generated by linking the two copies in a single sequence.

[0137] In some embodiments, the synthetic coiled-coil ERD of the disclosure comprises a chimeric sequence. Without being held to theory or mechanism, segments from ERDs capable of forming a coiled-coil structure may be grafted onto other ERDs capable of forming a coiled-coil structure to generate additional ERDs, if the grafting is in frame (i.e., the nucleic acid sequence aligns with the correct reading frame). Accordingly, a chimeric sequence may be formed by grafting a portion of an ERD capable of forming a coiled-coil structure onto another ERD capable of forming a coiled-coil structure, an example of which is depicted in FIG. 3. A chimeric sequence may also be formed by fusing, in frame, a portion of a first ERD capable of forming a coiled-coil structure to a portion of a second ERD capable of forming a coiled-coil structure.

[0138] In some embodiments, a chimeric sequence comprises a portion of a first ERD capable of forming a coiled-coil structure fused in frame to a portion of a second ERD capable of forming a coiled-coil structure.

[0139] In some embodiments, the portion of the first ERD fused in frame to a portion of a second ERD is at the N-terminus of the first ERD. In some embodiments, the portion of the first ERD fused in frame to a portion of a second ERD is at the C-terminus of the first ERD. In some embodiments, the portion of the first ERD fused in frame to a portion of a second ERD is in the middle of the first ERD.

[0140] In some embodiments, the portion of the first ERD fused in frame to a portion of a second ERD is about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 30%, about 1% to about 35%, about 1% to about 40%, about 1% to about 45%, about 1% to about 50%, about 1% to about 55%, about 1% to about 60%, about 1% to about 65%, about 1% to about 70%, about 1% to about 75%, or about 1% to about 80% of the total amino acid residues within the first ERD. In some embodiments, the portion of the first ERD fused in frame to a portion of a second ERD is about 1%, about 2%, about 3%, about 4 %, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total amino acid residues within the first ERD.

[0141] In some embodiments, the portion of the second ERD fused in frame to a portion of a first ERD is at the N-terminus of the second ERD. In some embodiments the portion of the second ERD fused in frame to a portion of a first ERD is at the C-terminus of the second ERD. In some embodiments, the portion of the second ERD fused in frame to a portion of a first ERD is in the middle of the second ERD.

[0142] In some embodiments, the portion of the second ERD fused in frame to a portion of a first ERD is about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 30%, about 1% to about 35%, about 1% to about 40%, about 1% to about 45%, about 1% to about 50%, about 1% to about 55%, about 1% to about 60%, about 1% to about 65%, about 1% to about 70%, about 1% to about 75%, or about 1% to about 80% of the total amino acid residues within the second ERD. In some embodiments, the portion of the second ERD fused in frame to a portion of a first ERD is about 1%, about 2%, about 3%, about 4 %, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total amino acid residues within the second ERD.

[0143] In some embodiments, a chimeric sequence is formed by replacing a portion of the sequence of a first ERD capable of forming a coiled-coil structure with a sequence derived from a second ERD capable of forming a coiled-coil structure.

[0144] In some embodiments, the portion of the sequence of the first ERD that is replaced is at the N-terminus of the first ERD. In some embodiments, the portion of the sequence of thefirst ERD that is replaced is at the C-terminus of the first ERD. In some embodiments, the portion of the sequence of the first ERD that is replaced is in the middle of the first ERD.

[0145] In some embodiments, the portion of the sequence of the first ERD that is replaced is about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 30%, about 1% to about 35%, about 1% to about 40%, about 1% to about 45%, about 1% to about 50%, about 1% to about 55%, about 1% to about 60%, about 1% to about 65%, about 1% to about 70%, about 1% to about 75%, or about 1% to about 80% of the total amino acid residues within the first ERD. In some embodiments, the portion of the sequence of the first ERD that is replaced is about 1%, about 2%, about 3%, about 4 %, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total amino acid residues within the first ERD.

[0146] In some embodiments, the sequence derived from the second ERD is at the N- terminus of the second ERD. In some embodiments, the sequence derived from the second ERD is at the C-terminus of the second ERD. In some embodiments, the sequence derived from the second ERD is in the middle of the second ERD.

[0147] In some embodiments, the sequence derived from the second ERD is about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 30%, about 1% to about 35%, about 1% to about 40%, about 1% to about 45%, about 1% to about 50%, about 1% to about 55%, about 1% to about 60%, about 1% to about 65%, about 1% to about 70%, about 1% to about 75%, or about 1% to about 80% of the total amino acid residues within the second ERD. In some embodiments, the sequence derived from the second ERD is about 1%, about 2%, about 3%, about 4 %, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total amino acid residues within the second ERD.

[0148] In some embodiments, the first ERD and / or the second ERD is a non-human homolog ERD. In certain embodiments, the non-human homolog ERD comprises the sequence of any one of SEQ ID NOs: 119-136 or an amino acid sequence having at least 70% sequence identity thereto. In some embodiments, the first ERD and / or the second ERD comprises the sequence of any one of SEQ ID NOs: 137-179, 252, 301-309, and 313-348 or an amino acid sequence having at least 70% sequence identity thereto.

[0149] Exemplary amino acid sequences of synthetic coiled-coil ERDs comprising a chimeric sequence are provided as SEQ ID NO: 348 and SEQ ID NO: 353. In certain embodiments, a synthetic coiled-coil ERD of the disclosure comprises the amino acid sequence of SEQ ID NO: 348 or SEQ ID NO: 353 or an amino acid sequence having at least 70% sequence identity thereto.

[0150] In some embodiments, the synthetic coiled-coil ERD described herein comprises any one of the amino acid sequences in Table 4A or Table 4B or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0151] In some embodiments, the synthetic coiled-coil ERD described herein comprises the sequence of any one of SEQ ID NOs: 137-179, 301-309, and 313-353, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto. In some embodiments, the synthetic coiled-coil ERD described herein comprises a truncated sequence of any one of SEQ ID NOs: 137-179, 301-309, and 313-353.

[0152] In some embodiments, said truncated sequence comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N terminus and / or C terminus of any one of SEQ ID NOs: 137-179, 301-309, and 313-353. In certain embodiments, said truncated sequence comprises a truncation of 9 or 10 amino acids from the N terminus and / or C terminus of any one of SEQ ID NOs: 137-179, 301-309, and 313-353. In some embodiments, said truncated sequence comprises a truncation of 9 or 10 amino acids from the N terminus and / or C terminus of any one of SEQ ID NOs: 137-179, 301-309, and 313-353. Without being held to theory or mechanism, truncation of a sequence may facilitate display of a single polypeptide construct with two synthetic ERD sequences, each comprising one sequence capable of forming an alpha helix. In some embodiments, the synthetic ERD described herein comprises a first sequence and a second sequence, wherein the first and / or second sequence is a truncated sequence of any one of SEQ ID NOs: 137-179, 301-309, and 313-353.

[0153] In some embodiments, the synthetic coiled-coil ERD described herein comprises the sequence of any one of SEQ ID NOs: 244-250 and 349-353, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0154] Table 4A provides exemplary amino acid sequences for the synthetic ERDs described herein having a backbone capable of forming a coiled-coil structure.Table 4A. Exemplary amino acid sequences of synthetic ERDs capable of forming coiled-coil structures

[0155] Table 4B provides exemplary amino acid sequences for synthetic ERDs described herein having a backbone capable of forming a coiled-coil structure. Each sequence in Table 4B comprises a first sequence capable of forming an alpha helix and a second sequence capable of forming an alpha helix, linked by a linker. The linker is optionally flanked by a proline at either end to facilitate breaking of the alpha helical structure. Although the sequences of Table 4B may be shown to include linkers, these linkers are optional in nature and may be omitted or replaced by other suitable linkers.Table 4B, Exemplary amino acid sequences of synthetic ERDs capable of forming coiled-coil structures

[0156] Also disclosed herein are polypeptides comprising a synthetic coiled-coil ERD described herein. It can be appreciated that the synthetic coiled-coil ERDs described herein may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, further discussed below in detail. Provided herein are polypeptides comprising a sequence in Table 4A or Table 4B, or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto..

[0157] Additionally, disclosed herein are polynucleotides encoding the polypeptides comprising a synthetic coiled-coil ERD, and disclosed herein are polynucleotides encoding the fusion proteins comprising a synthetic coiled-coil ERD. In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprises an antigenic polypeptide described herein and a synthetic coiled-coil ERD. Provided herein are polynucleotides that encode any one or more of the sequences of Table 4A and Table 4B, or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.. Polynucleotides can be DNA orRNA (e.g., mRNA), as will be described in more detail herein.D. Synthetic ERDs with ERMs

[0158] Also disclosed herein are synthetic ERDs generated by combining two or more ESCRT-recruiting motifs (ERMs) into a single polypeptide, also referred to as synthetic ERDs with ERMs. In some embodiments, the synthetic ERDs are generated by combining 2, 3, 4, 5, 6, 7, 8, 9, or 10 ERMs into a single polypeptide. In some embodiments, the synthetic ERD is generated by combining 2 ERMs into a single polypeptide. In some embodiments, the synthetic ERD is generated by combining 3 ERMs into a single polypeptide. In some embodiments, the synthetic ERD is generated by combining 4 ERMs into a single polypeptide. In some embodiments, the synthetic ERDs comprise at least 2 ERMs. In some embodiments, the synthetic ERDs comprise at least 3 ERMs. In some embodiments, the synthetic ERDs comprise at least 4 ERMs. In some embodiments, the synthetic ERDs comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 ERMs. In some embodiments, the synthetic ERDs comprise 2 ERMs. In some embodiments, the synthetic ERDs comprise 3 ERMs. In some embodiments, the synthetic ERDs comprise 4 ERMs. In some embodiments, the synthetic ERDs comprise 5 ERMs. In some embodiments, the synthetic ERDs comprise 6 ERMs. In some embodiments, the synthetic ERDs comprise 7 ERMs. In some embodiments, the synthetic ERDs comprise 8 ERMs. In some embodiments, the synthetic ERDs comprise 9 ERMs. In some embodiments, the synthetic ERDs comprise 10 ERMs.

[0159] In some embodiments, the synthetic ERDs comprises at least two ERMs, wherein the ERMs bind to one or more proteins associated with ESCRT. In some embodiments, the one or more ESCRT proteins is TSG101, ALIX, or a NEDD4-like protein. In certain embodiments, the one or more ESCRT proteins is TSG101. In certain embodiments, the one or more ESCRT proteins is ALIX. In certain embodiments, the one or more ESCRT proteins is a NEDD4-like protein. In some embodiments, the at least two ERMs independently comprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL, LXXLF, PPXY, L(X)nLXXLXXL, FPIV, PYXE, YXXL, LYPDLSEI, PLPPV, YEIL, and PYKELYPL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5. In certain embodiments, the at least two ERMs comprise the same sequence. In certain embodiments, the at least two ERMs comprise different sequences. In some embodiments, the various ERMs included in the synthetic ERDs described herein are selected independently. For example, a synthetic ERD comprising four ERMs may comprise two ERMs with unique sequences and two ERMs with the same sequence. In some embodiments, an ERM comprises a sequence in Table 2B or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, atleast 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto. In some embodiments, an ERM comprises a sequence inTable 2A or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto. In some embodiments, an ERM comprises a sequence of any one of SEQ ID NOs: 4-15 and 354- 364, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0160] In some embodiments, a synthetic ERDs with ERMs described herein further comprises a linker. In some embodiments, the synthetic ERD comprises, from N-terminus to C- terminus:(a) a first ERM, a linker, and a second ERM;(b) a linker, a first ERM, a linker, a second ERM, and a linker;(c) a first ERM, a linker, a second ERM, a linker, and a third ERM;(d) a linker, a first ERM, a linker, a second ERM, a linker, a third ERM, and a linker;(e) a first ERM, a linker, a second ERM, a linker, a third ERM, a linker, and a fourth ERM; or(f) a linker, a first ERM, a linker, a second ERM, a linker, a third ERM, a linker, a fourth ERM, and a linker.

[0161] In some embodiments, the synthetic ERD comprises, from N-terminus to C- terminus: a first ERM, a linker, a second ERM, a linker, a third ERM, a linker, and a fourth ERM.

[0162] In some embodiments, the synthetic ERD comprises, from N-terminus to C- terminus: a linker, a first ERM, a linker, a second ERM, a linker, a third ERM, a linker, a fourth ERM, and a linker

[0163] The linker included in a synthetic ERD may be any linker described herein. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a Glycine- Serine (GS or SG) linker. In some embodiments, the linker comprises an amino acid sequence of SGG, SSG, GSG, GG, GSGG, NGTGGSG, GGGGS, or the like. In some embodiments, the linker is a rigid linker. In some embodiments, the linker comprises any one of the sequences in Table 8A or Table 8B. In some embodiments, the linker comprises the sequence of any one of SEQ ID NOs: 235-243.

[0164] Without being held to theory or mechanism, in some embodiments, arranging ESCRT-recruiting motifs (ERMs) in a certain order may improve eVLP production. For example, in some embodiments where the P(T / S)AP, PPXY, or YP(X)nL motifs come N terminal of another motif, such as the LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL or IYPVRSNSTI motif, ERD activity (e.g. eVLP production) is increased. As another example, in some embodiments, placing the P(T / S)AP motif(s) N terminal to the LXXLF, the LXXLXXL, the LYPDLSEI, PYKELYPL, or the IYPVRSNSTI motifs results in increased ERD activity. In yet another example, in some embodiments, increased ERD activity results from placing the LXXLF, the LXXLXXL, the LYPDLSEI, PYKELYPL, or the IYPVRSNSTI motifs C terminal to another motif. Unless otherwise indicated, the letter “X” in any of the motifs described herein denotes any amino acid and the letter “n” denotes 1, 2, 3, 4, or 5.

[0165] In some embodiments, the synthetic ERDs described herein comprise an ESCRT- recruiting motif (ERM) that binds to a NEDD4-like protein and an ERM that binds to ALIX, wherein the ERM that binds to a NEDD4-like protein is N-terminal to the ERM that binds to ALIX.

[0166] In some embodiments of the synthetic ERDs described herein, an ERM motif of P(T / S)AP, PPXY, or YP(X)nL comes N terminal to an ERM motif of LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI, wherein x is any amino acid and n is 1, 2, 3, 4, or 5. In some embodiments, the synthetic ERDs described herein comprise a first ERM comprising the sequence P(T / S)AP, PPXY, or YP(X)nL and a second ERM comprising the sequence LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI, wherein said first ERM is N- terminal to the second ERM, and wherein X is any amino acid and n is 1, 2, 3, 4, or 5.

[0167] In some embodiments, an ERM included in the synthetic ERDs described herein comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91%sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0168] In some embodiments, the synthetic ERDs with ERMs described herein comprise the amino acid sequence of any one of SEQ ID NOs: 180-234 and 365-367, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0169] Table 4C provides exemplary amino acid sequences for the synthetic ERDs with ESCRT-recruiting motifs (ERMs) described herein. Although the sequences of Table 4C are shown to include Glycine-Serine (GS or SG) linkers, these linkers may be replaced by other suitable linkers.Table 4C. Exemplary Amino Acid Sequences of Synthetic ERDs containing ESCRT -recruiting motifs

[0170] Also disclosed herein are polypeptides comprising a synthetic ERD with ERMs described herein. It can be appreciated that the synthetic ERD with ERMs described herein may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, further discussed below in detail. Provided herein are polypeptides comprising a sequence in Table 4C, or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto..

[0171] Additionally, disclosed herein are polynucleotides encoding the polypeptides comprising a synthetic ERD with ERMs, and disclosed herein are polynucleotides encoding the fusion proteins comprising a synthetic ERD with ERMs. In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprises an antigenic polypeptide described herein and a synthetic ERD with ERMs. Provided herein are polynucleotides that encode any one or more of the sequences of Table 4C, or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequenceidentity, or at least 99% sequence identity thereto. Polynucleotides can be DNA or RNA (e.g., mRNA), as will be described in more detail herein.E, Tandem ERDs

[0172] In some instances, it may be desirable or advantageous to enhance engagement of an ERD sequence to ESCRT proteins (e.g. TSG101 and / or ALIX). To enhance engagement of ESCRT proteins (e.g. TSG101, ALIX, and / or NEDD4), two or more copies of an ERD sequence may be included in tandem in a polypeptide or a fusion protein described herein. An ERD comprising two or more copies of an ERD sequence described herein is referred to as a tandem ERD. In some embodiments, a tandem ERD described herein comprises at least two ERDs. In some embodiments, the at least two ERDs both comprise a sequence which is capable of forming the same structure. Non-limiting examples of such structures include a linear polypeptide chain, an alpha helix, or a coiled-coil structure. For example, in certain embodiments, a tandem ERD comprises two ERDs, each comprising a sequence capable of forming alpha helix. Without being held to theory or mechanism, the

[0173] In some embodiments, a tandem ERD described herein comprises a first ERD and a second ERD. In certain embodiments, the first and the second ERD of a tandem ERD may be linked by a linker. In some embodiments, the first ERD and the second ERD comprises the same amino acid sequence. In some embodiments, the first ERD and the second ERD comprises different amino acid sequences.

[0174] A tandem ERD described herein which comprises a first viral ERD and a second viral ERD are referred to as a viral tandem ERD. In some embodiments, a viral tandem ERD comprises two copies of the same viral ERD. In some embodiments, a viral tandem ERD comprises two different viral ERDs. In some embodiments, the two copies of the viral ERD are isolated or derived from the same virus. In some embodiments, the two copies of the viral ERD are isolated or derived from two different viruses. In some embodiments, the first virus and / or the second virus is selected from the group consisting of: equine infectious anemia virus (EIAV), human immunodeficiency virus (HIV), Ebola (EBOV), Mason-Pfizer monkey virus (MPMV), Human T-lymphotropic virus (HTLV), vaccinia virus (Vacc.), tick-borne encephalitis virus (TBEV), simian virus (SV-5), rous sarcoma virus, porcine endogenous retrovirus (PERV), mouse mammary tumor virus (MMTV), prototype foamy virus (PFV), feline immunodeficiency virus (FIV), lymphocytic choriomeningitis virus (LCMV), PEAV (porcine enteric alphacoronavirus), simian immunodeficiency virus (SIV), human metapneumovirus (hMPV),human T Cell Leukemia Virus, Bovine Foamy Virus, mumps virus, vesicular stomatitis virus (VSV), and Mammarenavirus lassaense.

[0175] Without being held to theory or mechanism, in some embodiments, arranging ERDs in a certain order may improve eVLP production. For example, in some embodiments, placing an ERD derived from MPMV, EIAV, EBOV, or HIV on the C terminal end of a tandem ERD may lead to increased ERD activity. In another example, in some embodiments, placing an ERD derived from vaccinia virus or HTLV-1 leads to decreased ERD activity. In some embodiments, the first sequence derived from the first virus and / or the second sequence derived from the second virus comprises an ESCRT-recruiting motif (ERM). In some embodiments, the ERM binds, engages, or recruits one or more ESCRT proteins (e.g. TSG101, ALIX, and / or NEDD4). In some embodiments, the one or more ESCRT proteins is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4-like protein. In certain embodiments, the one or more ESCRT proteins is TSG101. In certain embodiments, the one or more ESCRT proteins is ALIX. In certain embodiments, the one or more ESCRT proteins is a NEDD4-like protein.

[0176] In some embodiments, the first and / or the second ERD of the tandem ERDs described herein comprises at least one, at least two, at least three, or at least four ERM. In some embodiments, the first and / or the second ERD of the tandem ERDs described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ERMs. In certain embodiments, the first and / or the second ERD comprises 1 ERM. In certain embodiments, the first and / or the second ERD comprises 2 ERMs. In certain embodiments, the first and / or the second ERD comprises 3 ERMs. In certain embodiments, the first and / or the second ERD comprises 4 ERMs. In some embodiments, the ERM comprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL, LXXLF, PPXY, L(X)nLXXLXXL, FPIV, PYXE, YXXL, LYPDLSEI, PLPPV, YEIL, and PYKELYPL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5. In some embodiments, the ERM comprises a sequence in Table 2B. In some embodiments, the ERM comprises a sequence in Table 2A.

[0177] In certain embodiments, the ERM is derived from a Late Domain. In some embodiments, the first and / or second ERD of a tandem ERD disclosed herein comprises an ERM derived from a Late Domain. In some embodiments, the first and / or second ERD of a tandem ERD disclosed herein comprise a Late Domain. In some embodiments, the Late Domain binds, engages, or recruits one or more ESCRT proteins (e.g. TSG101, ALIX, and / or NEDD4). In some embodiments, the one or more ESCRT proteins is TSG101, ALIX, or a NEDD4-likeprotein. In certain embodiments, the one or more ESCRT proteins is TSG101. In certain embodiments, the one or more ESCRT proteins is ALIX. In certain embodiments, the one or more ESCRT proteins is a NEDD4-like protein.

[0178] Exemplary ERMs are provided in Table 2A and Table 2B. In some embodiments, the first and / or second ERD of a tandem ERD disclosed herein comprises an ERM comprising any one of the sequences in Table 2A or Table 2B, or a sequence with at least 70% sequence identity thereto.

[0179] In some embodiments, the first and / or second ERD of a tandem ERD disclosed herein comprises an ERM comprising the sequence of any one of SEQ ID NOs: 4-15 and 354- 364, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0180] In some embodiments, the tandem ERDs disclosed herein comprise an ERM comprising the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0181] In some embodiments, the first and / or the second ERD of a tandem ERD disclosed herein comprises the amino acid sequence of any one of SEQ ID NOs: 16-51 or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0182] In some embodiments, the first and the second ERD of a tandem ERD are linked by a linker. The linker may be any linker described herein. In some embodiments, the linker is a flexible linker. In some embodiments, the flexible linker is a Glycine-Serine linker, also referred to as a GS or SG linker. In some embodiments, the linker comprises an amino acid sequence ofSGG, SSG, GSG, GG, GSGG, NGTGGSG, GGGGS, or the like. In some embodiments, the linker comprises any one of the sequences in Table 8 A or Table 8B.

[0183] In some embodiments, the tandem ERD described herein comprises any one of the sequences in Table 5. In some embodiments, the tandem ERD described herein comprises the amino acid sequence of any one of SEQ ID NOs: 52-116 or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0184] Table 5 provides exemplary sequences for the tandem ERDs described herein. Although the sequences of Table 5 may be shown to include linkers, these linkers are optional in nature and may be omitted or replaced by other suitable linkers.Table 5: Exemplary viral tandem ERD sequences

[0185] Also disclosed herein are polypeptides comprising a tandem ERD described herein. It can be appreciated that the tandem ERD described herein may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, further discussed below in detail. Provided herein are polypeptides comprising a sequence in Table 5, or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94%sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto..

[0186] Additionally, disclosed herein are polynucleotides encoding the polypeptides comprising a tandem ERD, and disclosed herein are polynucleotides encoding the fusion proteins comprising a tandem ERD. In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprises an antigenic polypeptide described herein and a tandem ERD. Provided herein are polynucleotides that encode any one or more of the sequences of Table 5, or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.. Polynucleotides can be DNA or RNA (e.g., mRNA), as will be described in more detail herein.

[0187] Table 6 provides exemplary amino acid sequences for an ERD comprising a truncated human EABR sequence (EABR.WT. Short) and an ERD comprising an engineered EABR comprising mutations relative to wild type EABR sequences (EABR. AllMut). These ERDs may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein.Table 6, Other exemplary ERD sequences

[0188] Provided herein are polynucleotides that encode any one or more of the sequences of Table 6, or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto. Polynucleotides can be DNA or RNA (e.g., mRNA), as will be described in more detail herein. Also provided herein arepolypeptides comprising a sequence in Table 6, or a sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto. Also disclosed herein are polypeptides encoded by any one of the polynucleotides described herein.

[0189] In some embodiments, an ERD of the disclosure recruits and / or binds to an ESCRT protein, such as Alix, TSG101, and / or NEDD-4. Accordingly, provided herein are methods to test the binding of an ERD of the disclosure to an ESCRT protein. In some embodiments, an ERD of the disclosure is fused to a transmembrane domain of a human protein and expressed on the surface of a mammalian cell line. In certain embodiments, an ERD of the disclosure is fused to the transmembrane domain of human PDGFR protein. In some embodiments, a tag flanking the N terminus and / or the C terminus of the ERD is used as a marker for expression. In certain embodiments, the tag is a FLAG tag. In certain embodiments, the tag is a c-myc tag. In specific embodiments, a FLAG tag and a c-myc tag flanking the N terminus and C terminus of the ERD, respectively, are used as a marker for expression. In certain embodiments, the FLAG tag comprises the amino acid sequence EQKLISEEDL. In certain embodiments, the c-myc tag comprises the amino acid sequence DYKDDDDK. Any suitable cell line such as HEK293 cells, can then be transfected with such tagged constructs. Transfected cells can then be incubated with Alix, TSG101, or NEDD-4 proteins or peptides conjugated to fluorescently labeled streptavidin or directly fluorescently labeled. For example, cells can be incubated with Alix peptides conjugated Alexa Fluor 647 streptavidin and / or TSG101 peptides conjugated PE streptavidin. Transfected cells can then be stained with antibody specific to the FLAG or c-myc tag, followed by a fluorescently labeled secondary antibody that binds to FLAG or c-myc tag antibody, and analyzed via flow cytometry. Mock transfected cells can be used as a control to set the threshold for background fluorescence signals and guide the determination of the distribution of ERD-expressing cells. For example, the percentage of a population of transfected cells expressing ERDs that bind an ESCRT protein such as Alix, TSG101, and / or NEDD-4 can be quantified. In some embodiments, binding to Alix of transfected cells expressing ERDs is determined. In some embodiments, binding to TSG101 of transfected cells expressing ERDs is determined. In some embodiments, binding to NEDD-4 of transfected cells expressing ERDs is determined. In some embodiments, binding toAlix and TSG101 of transfected cells expressing ERDs is determined. In some embodiments, binding to Alix and NEDD-4 of transfected cells expressing ERDs is determined. In some embodiments, binding to NEDD-4 and TSG101 of transfected cells expressing ERDs is determined. In some embodiments, binding to Alix, TSG101, and NEDD-4 of transfected cells expressing ERDs is determined. In some embodiments, the distribution of ERD-expressing cells is visualized on a dot plot or histogram and / or quantified. For example, when shown on a scatter plot with fluorescence signal of Alix on one axis and fluorescence signal of TSG101 on the other axis, the percentage of (1) double negative cells, (2) TSG101 positive and Alix negative cells, (3) Alix positive and TSG101 negative cells, and (4) Alix / TSGlOl double positive cells can be quantified using mock transfected cells as a control. Alternatively, the percentage of binding of Alix or TSG101 positive cells relative to c-myc or flag positive cells can be used compared to mock transfected cells (c-myc negative control) or cells transfected with an off- target ERD (c-myc positive but Alix or TSG101 negative control). Other methods known in the art for assessing protein-protein interaction, such as surface plasmon resonance, may also be used to test, visualize, or quantify binding of an ERD to an ESCRT protein.Fusion Proteins

[0190] In another aspect, disclosed herein are fusion proteins comprising an antigenic polypeptide and an ERD described herein. Without being bound to theory or mechanism, the ERDs of the fusion proteins of the disclosure facilitate ESCRT protein recruitment and allow for spontaneous production of enveloped particles referred to herein as enveloped virus-like particles (eVLP) wherein the antigenic polypeptides (or part of the antigenic polypeptides) are displayed on the surface of the eVLPs. Virus-like particles (VLPs) are non-infectious particles whose production is driven by a viral capsid protein. Without being held to theory or mechanism, the eVLPs of the disclosure are non-infectious membraned particles whose production does not require a viral capsid protein and is instead driven by an ERD described herein, which is capable of recruiting a cell’s ESCRT machinery. ERDs of the disclosure are described in detail herein.

[0191] In some embodiments, the fusion proteins comprise a viral ERD, a non-human homolog ERD, a synthetic coiled-coil ERD, a synthetic ERD with ERMs, or a tandem ERD. In some embodiments, the fusion proteins comprise a viral ERD. In some embodiments, the fusion proteins comprise a non-human homolog ERD. In some embodiments, the fusion proteinscomprise a synthetic coiled-coil ERD. In some embodiments, the fusion proteins comprise a synthetic ERD with ERMs. In some embodiments, the fusion proteins comprise a tandem ERD.

[0192] In some embodiments, the fusion protein comprises one or more ERDs of the disclosure including one or more ERDs comprising an amino acid sequence in Table 2A, Table 2B, Table 2C, Table 3A, Table 3B, Table 4A, Table 4B, Table 4C, Table 5, or Table 6, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0193] In some embodiments, the fusion protein comprises one or more ERDs comprising an amino acid sequence of SEQ ID NOs: 4-116, 119-234, 244-250, 301-309, 313-353, or 365- 367, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0194] In some embodiments, a fusion protein disclosed herein comprises at least one antigenic polypeptide described herein. In some embodiments, the at least one antigenic polypeptide is derived from one of the exemplary antigens in Table 7A. In some embodiments, a fusion protein disclosed herein comprises an antigenic polypeptide. In some embodiments, the fusion protein of the disclosure comprises (i) an amino acid sequence of SEQ ID NOs: 1-3, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto, and (2) one or more ERD sequences, wherein the one or more ERD sequences comprises an amino acid sequence of SEQ ID NOs: 4-116, 119-234, 244-250, 301-309, 313-353, or 365-367, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0195] In some embodiments of the fusion proteins disclosed herein, an antigenic polypeptide is coupled to one or more ERD sequences described herein by a linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a Glycine-Serine (GS or SG) linker. In some embodiments, the linker sequence comprises an amino acid sequence SGG, GSG, GG, GSGG, NGTGGSG, GGGGS, or SGG, or any variation thereof. In some embodiments, the linker sequence comprises any one of the sequences in Table 8A or Table 8B.Antigenic Polypeptides

[0196] Disclosed herein are antigenic polypeptides that may be used in the fusion proteins of the present disclosure. An antigenic polypeptide may be associated with a bacterial or viral infection in a subject. An antigenic polypeptide may be isolated or derived from a bacterial or viral protein or a protein associated with a bacterial or viral infection.

[0197] In some embodiments, the antigenic polypeptides described herein are associated with a viral infection in a subject. In some embodiments, the antigenic polypeptides described herein comprise a polypeptide associated with a viral infection in a subject. In some embodiments, the antigenic polypeptides described herein are derived from a protein of a virus. An antigenic polypeptide described herein may be derived from a protein of a coronavirus, an Orthopoxvirus, a flavivirus, human metapneumovirus (hMPV), parainfluenza virus type 3 (PIV3), respiratory syncytial virus, varicella-zoster virus (VZV), cytomegalovirus (CMV), Herpes simplex virus (HSV) 1, HSV2, Epstein-Barr virus (EBV), or influenza.

[0198] In some embodiments, the antigenic polypeptides described herein are derived from a protein of an Orthopoxvirus. In some embodiments, the antigenic polypeptides described herein are derived from a protein of a flavivirus.

[0199] In some embodiments, the antigenic polypeptides described herein are derived from a protein of a coronavirus. In some embodiments, the coronavirus is SARS-CoV. In certain embodiments, the antigenic polypeptides described herein are derived from the spike protein of SARS-CoV. In certain embodiments, the antigenic polypeptides described herein are derived from the RBD of SARS-CoV.

[0200] In some embodiments, the coronavirus is MERS-CoV. In certain embodiments, the antigenic polypeptides described are derived from the spike protein of MERS-CoV. In certainembodiments, the antigenic polypeptides described are derived from the receptor-binding domain (RBD) of MERS-CoV.

[0201] In some embodiments, the coronavirus is SARS-CoV-2. Variant strains of SARS- CoV-2 have emerged and may emerge during a pandemic or endemic of SARS-CoV-2 infection. Variant strains of SARS-CoV-2 may also emerge at times outside of a pandemic or endemic, for instance, seasonally. In some embodiments, the coronavirus is a variant strain of SARS-CoV-2. In certain embodiments, the antigenic polypeptides described herein are derived from the spike protein of SARS-CoV-2 or variant strain thereof. In certain embodiments, the antigenic polypeptides described herein are derived from the RBD of SARS-CoV-2 or variant strain thereof.

[0202] In some embodiments, the antigenic polypeptides described herein are derived from a protein of hMPV. In certain embodiments, the antigenic polypeptides described herein are derived from the fusion (F) protein of hMPV.

[0203] In some embodiments, the antigenic polypeptides described herein are derived from a protein of PIV3. In certain embodiments, the antigenic polypeptides described herein are derived from the fusion (F) protein of PIV3.

[0204] In some embodiments, the antigenic polypeptides described herein are derived from a protein of respiratory syncytial virus. In certain embodiments, the antigenic polypeptides described herein are derived from the fusion (F) protein of respiratory syncytial virus.

[0205] In some embodiments, the antigenic polypeptides described herein are derived from a protein of VZV. In certain embodiments, the antigenic polypeptides described herein are derived from glycoprotein E (gE) of VZV.

[0206] In some embodiments, the antigenic polypeptides described herein are derived from a protein of CMV. In certain embodiments, the antigenic polypeptides described herein are derived from glycoprotein H (gH), glycoprotein L (gL), and / or glycoprotein B (gB) of CMV.

[0207] In some embodiments, the antigenic polypeptides described herein are derived from a protein of HSV1. In certain embodiments, the antigenic polypeptides described herein are derived from glycoprotein C (gH) and / or glycoprotein D (gL) of HSV1.

[0208] In some embodiments, the antigenic polypeptides described herein are derived from a protein of HSV2. In certain embodiments, the antigenic polypeptides described herein are derived from glycoprotein C (gH) and / or glycoprotein D (gL) of HSV2.

[0209] In some embodiments, the antigenic polypeptides described herein are derived from a protein of EBV.

[0210] In some embodiments, the antigenic polypeptides described herein are derived from a protein of influenza. In certain embodiments, the antigenic polypeptides described herein are derived from the hemagglutinin (HA) protein or neuraminidase (NA) protein of an influenza virus. In certain embodiments, the antigenic polypeptides described herein are derived from the head and / or stem regions of an influenza HA protein. In certain embodiments, the influenza virus is an influenza A virus. In certain embodiments, the influenza virus is an influenza B virus. In certain embodiments, the influenza virus is a seasonal influenza virus.

[0211] In some embodiments, the antigenic polypeptides described herein are associated with a bacterial infection in a subject. In some embodiments, the antigenic polypeptides described herein comprise a polypeptide associated with a bacterial infection in a subject. In some embodiments, the antigenic polypeptides described herein are derived from a protein of a bacterium. The bacterium may be of the genus Borrelia, Escherichia, Staphylococcus, or Chlamydia. An antigenic polypeptide described herein may be derived from a protein of Borrelia burgdorferi, E. Coli, Staphylococcus aureus, or Chlamydia trachomatis. An antigenic polypeptide described herein may be associated with infection in a subject with Lyme disease, E. Coli, staph, or chlamydia.

[0212] Table 7A provides exemplary antigens from which the antigenic polypeptides of the disclosure may be derived.Table 7A: Exemplary Antigens

[0213] Table 7B provides sequences of for exemplary antigenic polypeptides of the disclosure.Table 7B: Exemplary antigenic polypeptide sequences

[0214] In some embodiments, an antigenic polypeptide described herein comprises an amino acid sequence derived from any one of the antigens listed in Table 7A. Amino acid sequences for these antigens are publicly available in multiple databases, such as NCBI's Protein database, UniProt, and GenBank. Nucleic acid sequences for the antigens provided in Table 7A are publicly available in databases such as GenBank and RefSeq. In some embodiments, the antigenic polypeptide comprises an amino acid sequence derived from any one of the antigens listed in Table 7A, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.For example, in some embodiments, the antigenic polypeptide comprises an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to a sequence derived from an antigen listed in Table 7A.

[0215] In some embodiments, the antigenic polypeptide comprises a sequence in Table 7B, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0216] In some embodiments, the antigenic polypeptide comprises the sequence of any one of SEQ ID NOs: 1-3, or an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.Linkers

[0217] The various motifs, domains, polypeptides, and / or proteins described herein may be linked to each other through any suitable linker. Non-limiting examples of linkers are provided throughout the disclosure and in Table 8A and Table 8B.

[0218] Table 8A provides exemplary amino acid sequences for flexible linkers.Table 8 A, Exemplary flexible linker sequences

[0219] Table 8B provides exemplary amino acid sequences for rigid linkers.Table 8B, Exemplary rigid linker sequencesPolynucleotides

[0220] In another aspect, disclosed herein are polynucleotides encoding one or more of the ERDs described herein. Also disclosed herein are polynucleotides encoding polypeptide comprising one or more of the ERDs described herein.

[0221] In some embodiments, the polynucleotides disclosed herein encode a viral ERD, a non-human homolog ERD, a synthetic coiled-coil ERD, a synthetic ERD with ERMs, or a tandem ERD. In certain embodiments, the polynucleotides disclosed herein encode a viral ERD. In certain embodiments, the polynucleotides disclosed herein encode a non-human homolog ERD. In certain embodiments, the polynucleotides disclosed herein encode a synthetic coiled- coil ERD. In certain embodiments, the polynucleotides disclosed herein encode a synthetic ERDwith ERMs. In certain embodiments, the polynucleotides disclosed herein encode a tandem ERD.

[0222] In some embodiments, the polynucleotides disclosed herein encode a polypeptide comprising a viral ERD, a non-human homolog ERD, a synthetic coiled-coil ERD, a synthetic ERD with ERMs, or a tandem ERD. In certain embodiments, the polynucleotides disclosed herein encode a polypeptide comprising a viral ERD. In certain embodiments, the polynucleotides disclosed herein encode a polypeptide comprising a non-human homolog ERD. In certain embodiments, the polynucleotides disclosed herein encode a polypeptide comprising a synthetic coiled-coil ERD. In certain embodiments, the polynucleotides disclosed herein encode a polypeptide comprising a synthetic ERD with ERMs. In certain embodiments, the polynucleotides disclosed herein encode a polypeptide comprising a tandem ERD.

[0223] Additionally, disclosed herein are polynucleotides encoding any one or more of the fusion proteins described herein, wherein the fusion proteins comprise an antigenic polypeptide and an ERD. Also disclosed herein are polynucleotides encoding any one or more of the polypeptides described herein. In some embodiments, the polynucleotides comprises DNA or RNA (including mRNA). In some embodiments, the polynucleotides of the disclosure may be administered to a subject as a vaccine composition to prevent a disease or disorder. Also provided herein are polynucleotides that encode any one or more of the sequences, antigens, or polypeptides in Table 2A, Table 2B, Table 2C, Table 3A, Table 3B, Table 4A, Table 4B, Table 4C, Table 5, Table 6, or Table 7B

[0224] Table 8C provides exemplary nucleic acid sequences for the polynucleotides encoding a fusion protein described herein, wherein the fusion protein comprises a fruit bat EABR sequence and an antigenic polypeptide derived from the indicated antigen.Table 8C. Exemplary nucleic acid sequences of fusion proteins with a fruit bat EABR

[0225] Table 8D provides exemplary nucleic acid sequences for the polynucleotides encoding a fusion protein described herein, wherein the fusion protein comprises a tandem ERD and an antigenic polypeptide derived from the indicated antigen.Table 8D, Exemplary nucleic acid sequences of fusion proteins

[0226] In some embodiments, the polynucleotide disclosed herein comprises any one of the sequences in Table 8C or Table 8D, or a nucleic acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0227] In some embodiments, the polynucleotide disclosed herein comprises the sequence of any one of SEQ ID NOs: 253-284, or a nucleic acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0228] 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. 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.

[0229] 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 -methylpseudouridine, 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 in 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.

[0230] In some embodiments, where the polynucleotide of the present disclosure is mRNA, the mRNA molecule can be synthesized through in vitro transcription of acorresponding DNA template molecule. For example, synthetic mRNA can be produced by in vitro transcription of a cDNA template, such as plasmid DNA (pDNA).

[0231] Also provided herein are polypeptides encoded by any one of the nucleic acid sequences in Table 8C or Table 8D, or a nucleic acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0232] Also provided herein are polypeptides encoded by the nucleic acid sequence of any one of SEQ ID NOs: 253-284, or a nucleic acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0233] In another aspect, provided herein are cell anchored forms of an antigenic polypeptide described herein. An ERD of the disclosure may be used with a cell anchored antigenic polypeptide described herein to form a fusion protein. Exemplary nucleic acid sequences for cell anchored antigenic polypeptides are provided as SEQ ID NOs: 285-300. Accordingly, in some embodiments, the polynucleotide disclosed herein comprises the sequence of any one of SEQ ID NOs: 285-300, or a nucleic acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.

[0234] Also provided herein are polypeptides encoded by the nucleic acid sequence of any one of SEQ ID NOs: 285-300, or a nucleic acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, atleast 96% sequence identity, at least 98% sequence identity, or at least 99% sequence identity thereto.Vectors, Cells, eVLPs

[0235] In another aspect, provided herein are vectors comprising a polynucleotide described herein or a plurality of the polynucleotides described herein. 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. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. 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 described herein.

[0236] In some embodiments, the vectors of the present disclosure may be administered to a subject as a vaccine composition to prevent a disease or disorder.

[0237] In yet another aspect, disclosed herein is a cell that express on its cell surface any one of the fusion proteins or antigenic polypeptides described herein. In some embodiments, the cell expresses on its cell surface a portion of a fusion protein or a portion of an antigenic polypeptide described herein.

[0238] In yet another aspect, disclosed herein are enveloped virus-like particles (eVLPs), generated using the fusion proteins described herein, that display any one or more of the antigenic polypeptides. Without being held to theory or mechanism, 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 ERDs described herein. In some embodiments, the eVLP display on its surface any one of the antigenic polypeptides described herein, including any portion thereof. The range of sizes of theeVLPs produced using the methods of the disclosure may be of a size ranging from about 10 nm to about 150 nm.Methods

[0239] Also disclosed herein are methods of treating or preventing a disease or disorder, or a symptom thereof, in a subject in need thereof. In some embodiments, the methods comprises administering to the subject in need thereof an effective amount of any one of the polynucleotides encoding any one of the fusion proteins of the disclosure, any eVLP of the disclosure, or any vector of the disclosure.

[0240] In some embodiments, the administration is in the context of vaccination, and used for the prevention of an infection. In some embodiments, the infection is a viral infection.Examples of viral infections include, but are not limited to, infection with a coronavirus, an Orthopoxvirus, a flavivirus, human metapneumovirus (hMPV), parainfluenza virus type 3 (PIV3), respiratory syncytial virus, varicella-zoster virus (VZV), cytomegalovirus (CMV), Herpes simplex virus (HSV) 1, HSV2, Epstein-Barr virus (EBV), or influenza. In some embodiments, the infection is a bacterial infection, for example, chlamydia, acne, staph, or Lyme disease.

[0241] In some embodiments, the administration is in the context of treatment, and used for the mitigation of an already existing infection, and facilitates antibody-mediated clearance of the infection. In some embodiments, the infection is a viral infection. Examples of viral infection include, but are not limited to, a EBV infection, MERS infection, influenza infection, flavivirus infection, orthopoxvirus infection, or coronavirus infection.

[0242] The route of administration may be selected from any known method suitable for the treatment. In some embodiments, the route of administration is intravenous. In some embodiments, the route of administration is intramuscular. In some embodiments, the route of administration is subcutaneous.

[0243] In some embodiments, the subject in need thereof 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 non-human mammalian subject. In some embodiments, the subject is a non-human primate. In someembodiments, the subject is a llama. In some embodiments, the subject is a camel. In some embodiments, the subject is a cow.

[0244] In some embodiments, the subject is a bird. Non-limiting examples of birds include a chicken, a duck, a pheasant, a turkey, or a goose. In some embodiments, the subject is a chicken.

[0245] 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 viral or bacterial infection or is at risk of being infected by a virus or bacterium. In certain embodiments, the subject has had one or more prior infections with said virus or bacterium. In some embodiments, the subject has never been infected with said virus or bacterium.

[0246] The disclosure is 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

[0247] Provided herein are non-limiting exemplary enumerated embodiments.

[0248] Embodiment 1-1. A polynucleotide encoding a polypeptide, wherein the polypeptide comprises a viral endosomal sorting complex required for transport (ESCRT)- recruiting domain (ERD), wherein the viral ERD comprises a synthetic sequence derived from a virus.

[0249] Embodiment 1-2. The polynucleotide of embodiment 1, wherein the virus is selected from the group consisting of: equine infectious anemia virus (EIAV), human immunodeficiency virus (HIV), Ebola (EBOV), Mason-Pfizer monkey virus (MPMV), Human T-lymphotropic virus (HTLV), vaccinia virus (Vacc.), tick-borne encephalitis virus (TBEV), simian virus (SV-5), rous sarcoma virus, porcine endogenous retrovirus (PERV), prototype foamy virus (PFV), and feline immunodeficiency virus (FIV).

[0250] Embodiment 1-3. The polynucleotide of embodiment 1 or 2, wherein the sequence derived from the virus comprises an ESCRT -recruiting motif (ERM).

[0251] Embodiment 1-4. The polynucleotide of embodiment 3, wherein the ERM binds one or more ESCRT proteins.I l l

[0252] Embodiment 1-5. The polynucleotide of embodiment 4, wherein the one or more ESCRT proteins is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4-like protein.

[0253] Embodiment 1-6. The polynucleotide of any one of embodiments 3-5, wherein the ERM comprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL, LXXLF, PPXY, LXXLXXL, L(X)nLXXLXXL, PYXE, and YXXL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5.

[0254] Embodiment 1-7. The polynucleotide of any one of embodiments 3-6, wherein the ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto.

[0255] Embodiment 1-8. The polynucleotide of any one of embodiments 1-7, wherein the viral ERD comprises the amino acid sequence of any one of SEQ ID NOs: 16-51 or an amino acid sequence having at least 70% sequence identity thereto.

[0256] Embodiment 1-9. A polynucleotide encoding a polypeptide, wherein the polypeptide comprises a tandem endosomal sorting complex required for transport (ESCRT)- recruiting domain (ERD), wherein the tandem ERD comprises at least two ERDs.

[0257] Embodiment I- 10. The polynucleotide of embodiment 9, wherein the tandem ERD comprises a first ERD and a second ERD, wherein the first ERD comprises a first sequence derived from a first virus and the second ERD comprises a second sequence derived from a second virus.

[0258] Embodiment 1-11. The polynucleotide of embodiment 10, wherein the first and / or second virus is selected from the group consisting of: equine infectious anemia virus (EIAV), human immunodeficiency virus (HIV), Ebola (EBOV), Mason-Pfizer monkey virus (MPMV), Human T-lymphotropic virus (HTLV), vaccinia virus (Vacc.), tick-borne encephalitis virus (TBEV), simian virus (SV-5), rous sarcoma virus, porcine endogenous retrovirus (PERV), prototype foamy virus (PFV), and feline immunodeficiency virus (FIV).

[0259] Embodiment 1-12. The polynucleotide of embodiment 10 or 11, wherein the first virus and the second virus are the same virus.

[0260] Embodiment 1-13. The polynucleotide of embodiment 10 or 11, wherein the first virus and the second virus are not the same virus.

[0261] Embodiment 1-14. The polynucleotide of any one of embodiments 10 - 13, wherein the first and / or second sequence comprises an ESCRT -recruiting motif (ERM).

[0262] Embodiment 1-15. The polynucleotide of embodiment 14, wherein the ERM binds one or more ESCRT proteins.

[0263] Embodiment 1-16. The polynucleotide of embodiment 15, wherein the one or more ESCRT proteins is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4-like protein.

[0264] Embodiment 1-17. The polynucleotide of any one of embodiments 14-16, wherein the ERM comprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL, LXXLF, PPXY, LXXLXXL, L(X)nLXXLXXL, PYXE, and YXXL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5.

[0265] Embodiment 1-18. The polynucleotide of any one of embodiments 14-17, wherein the ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto.

[0266] Embodiment 1-19. The polynucleotide of embodiment 14, wherein the first sequence derived from a virus comprises a first ERM and the second sequence derived from a virus comprises a second ERM; wherein the first ERM is N-terminal to the second ERM; wherein the first ERM is PTAP, PSAP, PPxY, or YP(X)nL and the second ERM is LXXLF, LXXLXXL, LYPDLSEI, or IYPVRSNSTI; and wherein X is any amino acid and n is 1, 2, 3, 4, or 5.

[0267] Embodiment 1-20. The polynucleotide of any one of embodiments 14 - 19, wherein the first and / or the second ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto.

[0268] Embodiment 1-21. The polynucleotide of any one of embodiments 9-20, wherein the tandem ERD comprises a linker.

[0269] Embodiment 1-22. The polynucleotide of embodiment 21, wherein the linker is a flexible linker.

[0270] Embodiment 1-23. The polynucleotide of embodiment 21, wherein the linker is a Glycine-Serine linker.

[0271] Embodiment 1-24. The polynucleotide of embodiment 22 or 23, wherein the linker comprises the sequence of any one of SEQ ID NOs: 235-241.

[0272] Embodiment 1-25. The polynucleotide of any one of embodiments 9-24, wherein the tandem ERD comprises the amino acid sequence of any one of SEQ ID NOs: 52-116 or an amino acid sequence having at least 70% sequence identity thereto.

[0273] Embodiment 1-26. A polynucleotide encoding a polypeptide, wherein the polypeptide comprises a non-human homolog endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), wherein the non-human homolog ERD comprises a truncation relative to a naturally occurring non-human ERD.

[0274] Embodiment 1-27. The polynucleotide of embodiment 26, wherein the truncation relative to a naturally occurring non-human ERD comprises a truncation of 15 or less amino acids to the N terminus and / or the C terminus of a motif responsible for binding to one or more ESCRT proteins.

[0275] Embodiment 1-28. The polynucleotide of embodiment 26 or 27, wherein the naturally occurring non-human ERD is derived from a non-human vertebrate.

[0276] Embodiment 1-29. The polynucleotide of embodiment 28, wherein the non-human vertebrate is selected from the group consisting of: chicken, toad, mouse, elephant shrew, sunbittern, crocodile, Burmese python, fruit bat, pond turtle, and slow loris.

[0277] Embodiment 1-30. The polynucleotide of embodiment 26, wherein the naturally occurring ERD is derived from a fish.

[0278] Embodiment 1-31. The polynucleotide of embodiment 30, wherein the fish is selected from the group consisting of: zebrafish, greater pipefish, Atlantic halibut, snake pipefish, milkfish, European plaice, broad-nosed pipefish, banded pipefish, and goldfish.

[0279] Embodiment 1-32. The polynucleotide of any one of embodiments 26-31, wherein the non-human homolog ERD comprises an amino acid sequence of SEQ ID NOs: 117-136, or an amino acid sequence having at least 70% sequence identity thereto, or a portion thereof.

[0280] Embodiment 1-33. A polynucleotide encoding a polypeptide, wherein the polypeptide comprises a synthetic endosomal sorting complex required for transport (ESCRT)- recruiting domain (ERD), wherein the synthetic ERD comprises at least two ESCRT-recruiting motifs (ERMs).

[0281] Embodiment 1-34. The polynucleotide of embodiment 33, wherein the at least two ERMs bind to one or more proteins associated with ESCRT.

[0282] Embodiment 1-35. The polynucleotide of embodiment 34, wherein the one or more proteins associated with ESCRT is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4-like protein.

[0283] Embodiment 1-36. The polynucleotide of any one of embodiments 33-35, wherein each ERM comprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL,LXXLF, PPXY, LXXLXXL, L(X)nLXXLXXL, PYXE, and YXXL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5.

[0284] Embodiment 1-37. The polynucleotide of any one of embodiments 33-36, wherein each ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto.

[0285] Embodiment 1-38. The polynucleotide of any one of embodiments 33-36, wherein the synthetic ERD further comprises a linker.

[0286] Embodiment 1-39. The polynucleotide of embodiment 38, wherein the synthetic ERD comprises, from N-terminus to C-terminus: a. a first ERM, a linker, and a second ERM; b. a linker, a first ERM, a linker, a second ERM, and a linker; c. a first ERM, a linker, a second ERM, a linker, and a third ERM; d. a linker, a first ERM, a linker, a second ERM, a linker, a third ERM, and a linker; e. a first ERM, a linker, a second ERM, a linker, a third ERM, a linker, and a fourthERM; or f. a linker, a first ERM, a linker, a second ERM, a linker, a third ERM, a linker, a fourth ERM, and a linker.

[0287] Embodiment 1-40. The polynucleotide of embodiment 38 or 39, wherein the linker is a flexible linker.

[0288] Embodiment 1-41. The polynucleotide of embodiment 40, wherein the linker is a Glycine-Serine (GS or SG) linker.

[0289] Embodiment 1-42. The polynucleotide of embodiment 41, wherein the GS linker comprises the sequence of any one of SEQ ID NOs: 235-241.

[0290] Embodiment 1-43. The polynucleotide of embodiment 38 or 39, wherein the linker is a rigid linker.

[0291] Embodiment 1-44. The polynucleotide of embodiment 43, wherein the rigid linker comprises the sequence of SEQ ID NO: 242 or SEQ ID NO: 243.

[0292] Embodiment 1-45. The polynucleotide of any one of embodiments 33-44, wherein the synthetic ERD comprises an ERM that binds to a NEDD4-like protein and an ERM that binds to ALIX, wherein the ERM that binds to a NEDD4-like protein is N-terminal to the ERM that binds to ALIX.

[0293] Embodiment 1-46. The polynucleotide of any one of embodiments 33-44, wherein the synthetic ERD comprises a first ERM comprising a sequence of PTAP, PSAP, PPXY, orYP(X)nL and a second ERM comprising a motif different from that of the first ERM, wherein said first ERM is N-terminal to said second ERM.

[0294] Embodiment 1-47. The polynucleotide of any one of embodiments 33-44, wherein the synthetic ERD comprises a first ERM comprising a sequence of LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI and a second ERM comprising a motif different from that of the first ERM, wherein said first ERM is C-terminal to said second ERM.

[0295] Embodiment 1-48. The polynucleotide of any one of embodiments 33-44, wherein the synthetic ERD comprises an ERM comprising a sequence of PTAP, PSAP, PPXY, or YP(X)nL and an ERM comprising a sequence of LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI, wherein said PTAP, PSAP, PPXY, or YP(X)nL sequence is N- terminal to said LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI sequence.

[0296] Embodiment 1-49. The polynucleotide of any one of embodiments 33-48, wherein the synthetic ERD comprises the amino acid sequence of any one of SEQ ID NOs: 180-234 and 365-367, or an amino acid sequence having at least 70% sequence identity thereto.

[0297] Embodiment 1-50. A polynucleotide encoding a polypeptide, wherein the polypeptide comprises a synthetic coiled-coil endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), wherein the synthetic coiled-coil ERD comprises an amino acid sequence capable of binding one or more proteins associated with ESCRT; and wherein the synthetic coiled-coil ERD comprises a backbone capable of forming a coiled-coil structure.

[0298] Embodiment 1-51. The polynucleotide of embodiment 50, wherein the one or more proteins associated with ESCRT is selected from the group consisting of: tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), and a NEDD4-like protein.

[0299] Embodiment 1-52. The polynucleotide of embodiment 51, wherein the one or more proteins associated with ESCRT is TSG101 and / or ALIX.

[0300] Embodiment 1-53. The polynucleotide of any one of embodiments 50-52, wherein the synthetic coiled-coil ERD is computationally designed based on the structure of the ESCRT - and ALIX-binding region (EABR) sequence from the human CEP55 protein.

[0301] Embodiment 1-54. The polynucleotide of any one of embodiments 50-53, wherein the synthetic coiled-coil ERD has increased binding affinity to one or more proteins associated with ESCRT relative to the EABR sequence from the human CEP55 protein.

[0302] Embodiment 1-55. The polynucleotide of any one of embodiments 50-54, wherein the backbone comprises an amino acid sequence capable of forming an alpha helix.

[0303] Embodiment 1-56. The polynucleotide of embodiment 55, wherein the alpha helix interacts with another alpha helix formed by an identical sequence to make a 2-stranded coiled- coil structure.

[0304] Embodiment 1-57. The polynucleotide of any one of embodiments 50-56, wherein the synthetic coiled-coil ERD comprises the amino acid sequence of any one of SEQ ID NOs: 140-179, 301-309, and 313-353, or an amino acid sequence having at least 70% sequence identity thereto.

[0305] Embodiment 1-58. The polynucleotide of any one of embodiments 50-54, wherein the backbone comprises a first animo acid sequence capable of forming an alpha helix and a second amino acid sequence capable of forming an alpha helix.

[0306] Embodiment 1-59. The polynucleotide of embodiment 58, wherein the first animo acid sequence capable of forming an alpha helix and the second amino acid sequence capable of forming an alpha helix are linked by a linker.

[0307] Embodiment 1-60. The polynucleotide of embodiment 59, wherein the linker links the C-terminus of the first animo acid sequence to the N-terminus of the second amino acid sequence.

[0308] Embodiment 1-61. The polynucleotide of embodiment 59 or 60, wherein the linker is a flexible linker.

[0309] Embodiment 1-62. The polynucleotide of embodiment 59 or 60, wherein the linker is a rigid linker.

[0310] Embodiment 1-63. The polynucleotide of any one of embodiments 59 - 62, wherein the length of the linker is about 1 / 5 to 1 / 3 of the number of amino acids of the first or second amino acid sequence capable of forming an alpha helix.

[0311] Embodiment 1-64. The polynucleotide of any one of embodiments 58-63, wherein the synthetic coiled-coil ERD comprises the amino acid sequence of any one of SEQ ID NOs: 244-250 and 349-353, or an amino acid sequence having at least 70% sequence identity thereto.

[0312] Embodiment 1-65. The polynucleotide of any one of embodiments 50-56, wherein the backbone comprises an amino acid sequence derived from yeast GCN4.

[0313] Embodiment 1-66. The polynucleotide of embodiment 65, wherein the amino acid sequence derived from yeast GCN4 comprises the sequence of any one of SEQ ID NOs: 137- 139 or an amino acid sequence having at least 70% sequence identity thereto.

[0314] Embodiment 1-67. The polynucleotide of any one of embodiments 50-56, wherein the synthetic coiled-coil ERD comprises a chimeric sequence, wherein the chimeric sequencecomprises a portion of a first ERD capable of forming a coiled-coil structure fused in frame to a portion of a second ERD capable of forming a coiled-coil structure.

[0315] Embodiment 1-68. The polynucleotide of embodiment 67, wherein the first ERD and / or the second ERD is a non-human homolog ERD.

[0316] Embodiment 1-69. The polynucleotide of embodiment 68, wherein the non-human homolog ERD comprises the sequence of any one of SEQ ID NOs: 119-136 or an amino acid sequence having at least 70% sequence identity thereto.

[0317] Embodiment 1-70. The polynucleotide of embodiment 67, wherein the first ERD and / or the second ERD comprises the sequence of any one of SEQ ID NOs: 137-179, 252, 301- 309, and 313-348, or an amino acid sequence having at least 70% sequence identity thereto.

[0318] Embodiment 1-71. The polynucleotide of embodiment 70, wherein the synthetic coiled-coil ERD comprises the amino acid sequence of SEQ ID NO: 348 or SEQ ID NO: 353 or an amino acid sequence having at least 70% sequence identity thereto.

[0319] Embodiment 1-72. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises:(a) an antigenic polypeptide; and(b) an endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), wherein the ERD is:(i) a viral ERD;(ii) a tandem ERD;(iii) a non-human homolog ERD;(iv) a synthetic ERD comprising at least two ERMs; or(v) a synthetic coiled-coil ERD.

[0320] Embodiment 1-73. The polynucleotide of embodiment 72, wherein the antigenic polypeptide is derived from a protein of a virus.

[0321] Embodiment 1-74. The polynucleotide of embodiment 73, wherein the virus is hMPV, PIV3, respiratory syncytial virus, VZV, CMV, HSV1, HSV2, EBV, a coronavirus, influenza, a flavivirus, or orthopoxvirus.

[0322] Embodiment 1-75. The polynucleotide of embodiment 74, wherein the coronavirus is MERS-CoV, SARS-CoV, or SARS-CoV-2.

[0323] Embodiment 1-76. The polynucleotide of embodiment 75, wherein the protein is spike protein and the virus is MERS-CoV.

[0324] Embodiment 1-77. The polynucleotide of embodiment 75, wherein the protein is spike protein and the virus is SARS-CoV.

[0325] Embodiment 1-78. The polynucleotide of embodiment 75, wherein the protein is spike protein and the virus is SARS-CoV-2.

[0326] Embodiment 1-79. The polynucleotide of embodiment 74, wherein the protein is fusion (F) protein and the virus is hMPV.

[0327] Embodiment 1-80. The polynucleotide of embodiment 74, wherein the protein is fusion (F) protein and the virus is PIV3.

[0328] Embodiment 1-81. The polynucleotide of embodiment 74, wherein the protein is fusion (F) protein and the virus is respiratory syncytial virus.

[0329] Embodiment 1-82. The polynucleotide of embodiment 74, wherein the protein is glycoprotein E (gE) and the virus is VZV.

[0330] Embodiment 1-83. The polynucleotide of embodiment 74, wherein the protein is glycoprotein H (gH), glycoprotein L (gL), or glycoprotein B (gB) and the virus is CMV.

[0331] Embodiment 1-84. The polynucleotide of embodiment 74, wherein the protein is glycoprotein C (gC) or glycoprotein D (gD) and the virus is HSV1 or HSV2.

[0332] Embodiment 1-85. The polynucleotide of embodiment 72, wherein the antigenic polypeptide is derived from a protein of a bacterium.

[0333] Embodiment 1-86. The polynucleotide of embodiment 85, wherein the bacterium is an acne-causing bacterium, Staphylococcus, Borrelia, E. Coli, or Chlamydia.

[0334] Embodiment 1-87. The polynucleotide of embodiment 86, wherein the protein is DsAl and the acne-causing bacterium is Cutibacterium acnes.

[0335] Embodiment 1-88. The polynucleotide of embodiment 86, wherein the protein is esxA or esxB and the bacterium is Staphylococcus.

[0336] Embodiment 1-89. The polynucleotide of embodiment 86, wherein the protein is OspA and the bacterium is Borrelia, optionally Borrelia burgdorferi.

[0337] Embodiment 1-90. The polynucleotide of embodiment 86, wherein the protein is FimH and the bacterium is E. Coli.

[0338] Embodiment 1-91. The polynucleotide of embodiment 86, wherein the protein is major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), or OmcB and the bacterium is Chlamydia.

[0339] Embodiment 1-92. The polynucleotide of any one of embodiments 72-91, wherein the ERD is a viral ERD comprising a sequence derived from a virus.

[0340] Embodiment 1-93. The polynucleotide of any one of embodiments 72-91, wherein the ERD is a tandem ERD comprising at least two ERDs.

[0341] Embodiment 1-94. The polynucleotide of any one of embodiments 72-91, wherein the ERD is a non-human homolog ERD.

[0342] Embodiment 1-95. The polynucleotide of any one of embodiments 72-91, wherein the ERD is a synthetic ERD comprising at least two ESCRT-recruiting motifs (ERMs).

[0343] Embodiment 1-96. The polynucleotide of any one of embodiments 72-91, wherein the ERD is a synthetic coiled-coil ERD, wherein the synthetic coiled-coil ERD comprises an amino acid sequence capable of binding one or more proteins associated with ESCRT, wherein the synthetic ERD comprises a backbone capable of forming a coiled-coil structure.

[0344] Embodiment 1-97. The polynucleotide of any one of embodiments 1-96, wherein the polynucleotide comprises RNA.

[0345] Embodiment 1-98. The polynucleotide of embodiment 97, wherein the polynucleotide comprises mRNA.

[0346] Embodiment 1-99. The polynucleotide of any one of embodiments 1-96, wherein the polynucleotide comprises DNA.

[0347] Embodiment I- 100. A polypeptide encoded by the polynucleotide of any one of embodiments 1-99.

[0348] Embodiment I- 101. A cell expressing on its surface the fusion protein, or a portion of the fusion protein, encoded by the polynucleotide of any one of embodiments 72-99.

[0349] Embodiment 1-102. An enveloped virus-like particle (eVLP) comprising the fusion protein encoded by the polynucleotide of any one of embodiments 72-99.

[0350] Embodiment 1-103. An enveloped virus-like particle (eVLP) displaying on its surface the antigenic polypeptide, or part of the antigenic polypeptide, of the fusion protein encoded by the polynucleotide of any one of embodiments 72-99.

[0351] Embodiment 1-104. The eVLP of embodiment 102 or 103, wherein the diameter of the eVLP is 10 nm - 150 nm.

[0352] Embodiment 1-105. A vector comprising the polynucleotide of any one of embodiments 1- 99.

[0353] Embodiment 1-106. The vector of embodiment 105, wherein the vector is a viral vector.

[0354] Embodiment 1-107. The vector of embodiment 105, wherein the vector is a non- viral vector.

[0355] Embodiment 1-108. The vector of embodiment 107, wherein the non-viral vector is a plasmid.

[0356] Embodiment 1-109. The vector of embodiment 107, wherein the non-viral vector is a lipid nanoparticle (LNP).

[0357] Embodiment 1-110. The vector of embodiment 109, wherein the non-viral vector is a lipid nanoparticle (LNP) and the polynucleotide comprises mRNA.

[0358] Embodiment 1-111. A method of preventing or treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of any one of the polynucleotides of any one of embodiments 72-99, the eVLP of any one of embodiments 102-104, or the vector of any one of embodiments 105-110.

[0359] Embodiment 1-112. The method of embodiment 111, wherein the subject is a human subject.EXAMPLESExample 1: Generation and Characterization of Non-Human ERDs using MERS Spike eVLPs

[0360] An investigation was conducted to profile a panel of non-human ESCRT recruiting domains (ERDs). ERDs, in a vaccine setting, aim to concomitantly enable expression of an antigen of interest on the cell surface and produce eVLPs in situ as a result of ESCRT recruitment. As a representative antigen, that could be linked to the ERDs under evaluation, a truncated MERS spike protein, termed MERS-1227 was employed. MERS-1227 is a derivative of the spike protein sourced from the Middle East respiratory syndrome coronavirus (MERS- CoV), which is truncated at position 1227. This antigen, in a variety of forms, including as an mRNA-delivered antigen with a transmembrane (TM) domain and C-terminal domain (CTD) is antigenic in mouse models. Accordingly, MERS-1227 with a TM domain and CTD (SEQ ID NO: 1), served as an representative model antigen for initial ERD evaluation.

[0361] A total of 84 non-human ERDs underwent evaluation in this study. Wildtype human CEP55 EABR (serving as the positive control) (SEQ ID NO: 252: FNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAAHSLP) and its truncated variant which was designed to retain the resolved portions from the crystal structure (EABR. WT. short; SEQ ID NO: 117) were included as reference points. Concurrently, a mock transfection (designated as Blank) and plasmid encoding the full-length, fusioncompetent, MERS spike protein devoid of ERD (EABR null control) served as the negative controls. For high-throughput screening, all ERD constructs were administered as plasmid DNA to both human cell line HEK293T and mouse cell line 3T3. Subsequently, 72-hours posttransfection, the levels of eVLPs produced from each construct in the supernatant were quantified via dot blot analysis, while the impact of ERDs on overall MERS spike expression levels was gauged through cell ELISA. MERS spike antibody D12 was employed as the detection antibody in both assays. All exemplary ERD sequences were positioned at the C- terminus of the exemplary MERS spike protein (SEQ ID NO: 1), which contained a transmembrane domain and a short cytoplasmic domain (as depicted in FIGS. 2A-2E). The polynucleotides used in the following assays encode a fusion protein comprising the antigenic polypeptide of any one of SEQ ID NOs: 1, 2, and 3, followed by any one of the ERDs named in Table 9A, the sequences of which are provided in Table 3.Table 9A: Plate layout comprising the exemplary ERD sequences

[0362] In the dot blot analysis conducted on transfected HEK293T cells, all non-human vertebrate EABRs (positioned vertically from BIO to Bl 1 in FIGS. 4A-4B) in addition to the positive control (G11 in FIGS. 4A-4B) exhibited robust ERD activity. In contrast, a varied range of ERD activity was observed with viral ERDs and synthetic ERDs based on GCN4. Notably, ERDs derived from vaccinia virus (ranging from Hl to H8 and A8, B8, etc. to G8 in FIGS. 4A-4B) emerged as the least performing, indicating that the vaccinia ERD sequence might impede eVLP formation. Because the MERS spike protein as a TM domain, for it to be detected in the cell supernatant it is presumed to have budded from the cell, while embedded in an eVLP, in an ESCRT-dependent fashion. Darker dots indicate more MERS spike protein is present in the sample and presumably that means more budding from the surface of the cell.

[0363] Although with diminished intensity, dot blots generated from ERD-transfected mouse cell line 3T3 (FIG. 5) exhibited a pattern akin to that of HEK293T cells. Results are functionally similar but with lower overall yield.

[0364] In the scatter plot depicting antigen expression level on the x-axis and ERD activity on the y-axis, it is noteworthy to observe that viral tandem ERDs showcased a wider dispersion compared to ERDs sourced from a single virus (depicted as viral solo in FIG. 6), spanning both x- and y-axes. This outcome underscores that fusing a viral ERD with another viral ERD may either dampen or enhance ERD activity, alongside the expression level of the antigen within the cells and that, surprisingly, tandem forms of ERDs can have improved ERD activity. Incorporating ERDs sourced from HIV, EIAV, and MPMV predominantly tended to enhance ERD activity, whereas integrating ERDs sourced from vaccinia virus often resulted in complete abrogation of the ERD activity inherent to the original ERD sequence.

[0365] To further delve into the eVLPs generated from diverse classes of ERDs scrutinized in this study, a selection of 12 ERDs spanning viral tandem, synthetic, and vertebrate categories (FIG. 7) were chosen for isolating eVLPs from transfected Expi293F cell culture.The intensity of bands observed in purified eVLPs (FIG. 10) mirrored that of the dot plot (FIG. 4B), implying that the dot intensity in dot blots accurately reflects the quantity of eVLPs generated by ERDs. The fusion proteins ran at the anticipated molecular weight as seen in FIG. 10. As seen by the gel, the purified eVLPs contain predominantly the MERS spike protein of interest with minimal host cell protein contamination.

[0366] The size of purified eVLPs was assessed via dynamic light scattering (DLS). eVLPs generated from diverse ERDs ranged from approx, radiuses of 40 to 80nm (FIG. 8). eVLPs were purified via an N-terminal Strep Tag 2 using StrepTactin® resin. ERD-based purified eVLPs show a range of sizes consistent with exosomes, suggesting the purified proteins are embedded in, or associated with, a lipid bilayer and entire eVLPs are being co-purified.Collectively, these data demonstrate that fusion proteins comprising the listed ERDs positioned C-terminus to an antigenic polypeptide (e.g., a MERS protein) are, in the context of genetic delivery (e.g. plasmid DNA) capable of producing the eVLPs as expected.

[0367] In comparison to viral and synthetic ERDs (exhibiting peaks ranging from 50 to 80nm), eVLPs induced by vertebrate ERDs appeared relatively smaller, with sizes ranging between 40 and 60nm. DLS results pertaining to individual ERDs can be found in FIGS. 9A to 9 J. The morphology of eVLPs originating from human CEP55 EABR was further confirmed through cryo-transmission electron microscopy (Cryo-TEM, FIG. 11).

[0368] To further demonstrate the applicability of ERDs, various classes of ERDs were fused to the C-terminus of gB and gp220 protein of Epstein-Barr virus (EBV). Like MERS- 1227, gB is a trimeric viral glycoprotein, whereas gp220 is a monomeric viral glycoprotein.Analogous to the MERS Spike protein, vertebrate EAB Rs emerged as the top performers for gB (Fig. 12A). However, discrepancies were noted between gB and gp220, such as the modest induction of gB eVLPs by EBOV.HIV ERD (#3 in FIG. 12A) contrasted with the absence of detectable eVLPs with gp220 (#3 in FIG. 12B). Conversely, MPMV.HIV ERD exhibited higher eVLP induction with gp220 compared to gB (#4 in FIG. 12A and FIG. 12B, respectively). Taken together, while some ERDs perform well with all antigens, the ERD that induces the desirable level of eVLP production may be influenced by characteristics of the antigen, such as its valency (monomeric, dimeric, trimeric, or tetrameric, etc.). For example, given that EABR and associated two-strand coil-coil sequences require a homodimer to function, EABRs may be better suited on dimeric / trimeric / tetrameric proteins, whereas viral ERDs or tandem viral ERDs may be similarly efficacious on monomeric proteins.Example 2: Generation and Characterization of Synthetic ERDs

[0369] As shown in Example 1, the ERD activity of a certain viral ERD can be significantly affected by the fusion of ERDs from the same or a different virus. To understand whether the location of an added ERD can affect the ERD activity of the original viral ERD, the effect of location was analyzed. As shown in FIG. 13, the addition of ERDs from MPMV, EIAV, EBOV, and HIV generally led to increase of ERD activity from the original ERD, whereas addition of ERDs from vaccinia virus and HTLV-1 generally resulted in decrease of ERD activity. The effect of adding SV5 and TBEV ERDs appeared minimal or unclear. For some cases, for example EIAV which has the LYPDLSEI motif, it was improved when EIAV ERD was put on the C terminal end. In another example, adding the viral ERD from HIV to the N terminus of the viral ERD from EBOV drastically increased EBOV ERD activity. However, adding the same ERD to the C terminus of the EBOV ERD led to a large decrease of ERD activity. These results imply that the order of ESCRT recruiting motifs within a viral ERD, a tandem viral ERD, and / or an engineered viral ERD may need to be placed in a particular order to have a higher level of ERD activity.

[0370] To test whether simply adding ESCRT recruiting motifs to a certain viral ERD would increase the activity of the original ERD, a total of 15 2ndGen ERDs were created by inserting ESCRT recruiting motifs to the ERD from MPMV.EIAV, one of the 1stGen viral tandem ERDs. Additionally, as several ESCRT recruiting motifs and their binding partner have been defined, it is possible to create fully synthetic viral ERDs by connecting ESCRT recruiting motifs with linkers. As shown in FIG. 15, four ESCRT-recruiting motifs (ERMs), PPPY, PSAP,PSAP, and PYKELYPL, derived from viral late domains were strung together into a single polypeptide sequence by a rigid linker (EAAAK)s (referred to as v2.16 in FIG. 14) or a flexible linker (GGSSGG)2 (referred to as v2.17 in FIG. 14). Each newly created ERD (referred to as 2nd Gen) was tested with and without an endocytosis prevention motif (EPM). As shown in FIG. 14, all the 2nd Gen ERDs displayed lower ERD activity compared to their parent sequences, which indicates that randomly inserting ESCRT recruiting motifs may lead to decrease of ERD activity. There was also no clear, consistent effect of EPM among different 2nd Gen constructs. ERD v2.17 shows an ERD activity close to ERD from MPMV.EIAV (referred to as 1stGen viral tandem ERD in FIG.14), supporting the concept that a fully synthetic ERD can be created by joining ESCRT recruiting motifs together with a flexible linker, such as a Glycine-Serine linker.

[0371] To determine the combinations of ESCRT recruiting motifs and the order by which these ESCRT recruiting motifs should be joined, a series of fully synthetic ERDs were designed by stringing together 4 ESCRT -recruiting motifs (ERMs) with (GGSSGG)2 linkers. Table 9B provides the fully synthetic ERDs (referred to as 3rd Gen ERDs) which were designed, with the ESCRT recruiting motifs used in each construct listed in order. In the ERD-informed constructs, which were designed based on the results shown in FIG. 13, a NEDD-4 binding motif was assigned to Motif 1 and an ALIX-binding motif, either LYPDLSEI or PYKELYPL, was assigned to Motif 4. A TSG101 -bindig motif or ERMs with unknown binding partner were randomly assigned to Motif 2 and Motif 3. In the random designs, ERMs were randomly assigned to each of the 4 motifs.Table 9B: Fully synthetic ERD constructs

[0372] ERD activity for the fully synthetic ERD constructs was measured by dot blot and cell ELISA using an anti-MERS D12 as the detection antibody. As shown in FIG. 16, ERD- informed designs showed not only much higher ERD activity (P < 0.0001) but also high expression level (P = 0.0430) than random ERD designs. While it is highly nonintuitive that the order in which the motifs are placed would play an important role in eVLP production given the motifs are identical, these results demonstrate that the order of the ERMs within an ERD should be designed and that following defined rules governing such order leads to ERDs with significantly higher ERD activity, with said ERDs engaging the essential host factors, such as those in the ESCRT pathway, to induce budding of eVLPs.Table 10: Map of synthetic ERDs tested on hMPV F protein

[0373] A series of ERD sequences were then tested on hMPV F protein. The map of the sequences tested is shown in Table 10 and aligned with the dot blot shown in FIG. 17. Constructs labeled with “PMPNN” comprise synthetic ERDs whose sequences were determined using computational methods and designed based the structure of EABR from human CEP55. The dot blot of FIG. 17 was analyzed using densitometry to determine the intensity of each dot. Higher values represent better secretion as eVLPs and the cell anchored and fully secreted forms were included as controls. As shown in FIG. 18, many synthetic ERDs were successful in inducing eVLP formation and secretion. Importantly, the synthetic forms derived as PMPNN were developed, unlike ESCRT recruiting motifs derived from viral late domains, to be structurally defined. Essentially, these forms, like the GCN4 mutants tested above, were designed to retain the presumed coil-coil structure of the human EABR, but has more minimal human sequences. The computational tools were utilized to redevelop the coiled-coil, but retain only some critical residues important for the coiled-coil’ s interaction with host ESCRT machinery, such as TSG101 and ALIX. This provides, like the GCN4 version, an orthogonal structure-informed method for producing synthetic ERD sequences.

[0374] Mice were then immunized with constructs containing ERD-tagged hMPV F antigens. Animals received either a single dose (“prime only”) or two sequential doses (“prime + boost”) and serum was collected after immunization. Binding titers were calculated as an EC50 from ELISAs conducted against purified antigen. As shown in FIG. 19, an approximately 9-fold improvement in binding titers can be seen in animals after a receiving two doses (prime and boost) of a construct with an ERD tag, specifically that from Fruit Bat EABR.Table 11: Map of synthetic ERDs tested on hMPV F protein

[0375] A series of ERD sequences were then tested on VZV gE protein. The map of the sequences tested is shown in Table 11 and aligned with the dot blot shown in FIG. 20.Constructs labeled with “PMPNN” comprise synthetic ERDs whose sequences were determined using computational methods and designed based the structure of EABR from human CEP55. The dot blot of FIG. 20 was analyzed using densitometry to determine the intensity of each dot. Higher values represent better secretion as eVLPs and the cell anchored and fully secreted forms were included as controls. As shown in FIG. 21, many synthetic ERDs were successful in inducing eVLP formation and secretion. Importantly, the synthetic forms derived as PMPNN were developed, unlike viral late domain-associated ESCRT recruiting motifs, to be structurally defined. Essentially, these forms, like the GCN4 mutants tested above, were designed to retain the presumed coil-coil structure of the human EABR, but has more minimal human sequences. The computational tools were utilized to redevelop the coiled-coil, but retain only some critical residues important for the coiled-coil’ s interaction with host ESCRT machinery, such as TSG101 and ALIX. This provides, like the GCN4 version, an orthogonal structure-informed method for producing synthetic ERD sequences.

[0376] Mice were then immunized with constructs containing ERD-tagged hMPV F antigens. Animals received either a single dose (“prime only”) or two sequential doses (“prime + boost”) and serum was collected after immunization. Binding titers were calculated as an EC50 from ELISAs conducted against purified antigen. As shown in FIG. 22, an approximately 7-foldimprovement in binding titers can be seen in animals after a receiving single dose of a construct with an ERD tag, namely the EABR sequence from a Fruit Bat.Example 3: Generation and Characterization of Synthetic Coiled-coil ERDs

[0377] PMPNN is used to modify human EABR sequence in order to generate new ERDs completely or partially without extended stretches of human sequence identity and retain the coiled-coil structure of human EABR. With this approach, 35 additional sequences were created using PMPNN (SEQ ID NOs: 301-309, 313-327, and 349-352). To determine the degree of sequence homology needed for binding to Alix and TSG101, which are two components of ESCRT machinery, an EABR sequence was added to two PMPNN designs with no human EABR homology, PMPNN.14 and .15 (SEQ ID NO: 153 and SEQ ID NO: 154, respectively). With these two base sequences, 20 sequences (SEQ ID NOs: 328-347) with different degrees of sequence homology to human EABR were designed using PMPNN.

[0378] For a human EABR to perform its function in the ESCRT pathway, the EABR forms a dimer. Normally, the dimerization of EABR is a spontaneous reaction in the natural cell pathway. However, such spontaneous dimerization may not happen when EABR is fused to a foreign protein, such as a viral or bacterial antigen. Therefore, single-chain EABR sequences were created by introducing a linker to connect two copies of EABR or two copies of EABR short sequences, named EABR SC (SEQ ID NO: 349) and EABR SC short (SEQ ID NO: 350), respectively.

[0379] To test the binding of each PMPNN design and single-chain EABRs to Alix and TSG101, they are fused to the transmembrane domain of human PDGFR protein and expressed on the surface of a mammalian cell line. FLAG tag (amino acid sequence DYKDDDDK) and c- myc tag (amino acid sequence EQKLISEEDL) flanking each novel EDN’s N terminus and C terminus, respectively, were used as a marker for expression. Cells were then transfected with these constructs and incubated with Alix peptides conjugated to Alex Fluor 647 streptavidin (Alix- Alexa Flour 647), TSG101 peptides conjugated PE streptavidin (TSG101-PE), and antibody specific to FLAG or c-myc tag, followed by a fluorescently labeled secondary antibody that binds to FLAG or c-myc tag antibody.

[0380] Specifically, 1 million cells transfected with PMPNN design or single-chain EABR constructs or mock transfected cells were stained with Alix- Alexa Flour 647, TSG101-PE, and mouse anti-c-myc tag antibody, followed by anti-mouse secondary FITC conjugate and live / dead staining with Zombie NIR dye. The binding of ERDs to Alix and TSG101 was thenmeasured by flow cytometry. As shown in FIG. 23, live cells were gated first by forward (FSC) and side scatter (SSC), then single cells by FSC area (FSC-A) and height (FSC-H). To further exclude dead cells, cells that showed high levels of staining for Zombie NIR (shown as Alexa Fluor 750 on the x-axis) were excluded from the further analyses. The expression of synthetic coiled-coil ERDs designed using PMPNN and single-chain EAB Rs was measured by the c-myc tag (c-myc FITC). Mock transfected cells were used as a control to set the quadrants on the Alix-TSGlOl scatter plot at the bottom of FIG. 23, with quadrant (Q) 4 showing Alix / TSGlOl double negative cells, Q3 showing TSG101 positive and Alix negative cells, QI showing Alix positive and TSG101 negative cells, and Q2 showing Alix / TSGlOl double positive cells. By combining the histogram and Alix-TSGlOl scatter plot of FIG. 23, the distribution of ERD- expressing cells (black dots) can be observed on the scatter plot relative to Alix and TSG101 binding.

[0381] Human EABR has been reported to bind both Alix and TSG101. Cells transfected with human EABR or mock transfected cells were stained for binding to Alix and TSG101 and analyzed via flow cytometry, as described above. As shown in FIG. 24, 99.5% of the cells transfected with human EABR were Alix and TSG101 double positive (Q2). Taken together, analysis via flow cytometry can be used to determine expression of an ERD sequence in transfected cells and to test binding of an ERD sequence to ESCRT proteins such as Alix and TSG101.

[0382] An additional analysis of a broader number of ERD sequences was conducted via flow cytometry. HEK293T cell transfected with salmon sperm DNA (Mock transfection) was used as a negative control. As shown in Table 12 below, single-chain EABR dimers (SEQ ID NO: 349 and 350) showed higher binding to Alix and TSG101 peptides compared to the monomeric form of EABR. Six of the NPMNN BP designs tested demonstrate positive binding to both peptides. Taken together, these results demonstrate that synthetic ERD sequences designed using PMPNN, as well as single-chain EABR dimers (EABR SC and EABR SC short), are able to bind to Alix and TSG101.Table 12: Percentage of cells transfected with ERD sequences binding to Alix and / or TSG101

[0383] 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.

[0384] 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.

[0385] 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.

Claims

CLAIMS1. A polynucleotide encoding a polypeptide, wherein the polypeptide comprises a viral endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), wherein the viral ERD comprises a synthetic sequence derived from a virus.

2. The polynucleotide of claim 1, wherein the virus is selected from the group consisting of: equine infectious anemia virus (EIAV), human immunodeficiency virus (HIV), Ebola (EBOV), Mason -Pfizer monkey virus (MPMV), Human T-lymphotropic virus (HTLV), vaccinia virus (Vacc.), tick-borne encephalitis virus (TBEV), simian virus (SV-5), rous sarcoma virus, porcine endogenous retrovirus (PERV), prototype foamy virus (PF V), and feline immunodeficiency virus (FIV).

3. The polynucleotide of claim 1 or 2, wherein the sequence derived from the virus comprises an ESCRT-recruiting motif (ERM).

4. The polynucleotide of claim 3, wherein the ERM binds one or more ESCRT proteins.

5. The polynucleotide of claim 4, wherein the one or more ESCRT proteins is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4- like protein.

6. The polynucleotide of any one of claims 3-5, wherein the ERM comprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL, LXXLF, PPXY, LXXLXXL, L(X)nLXXLXXL, PYXE, and YXXL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5.

7. The polynucleotide of any one of claims 3-6, wherein the ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto.

8. The polynucleotide of any one of claims 1-7, wherein the viral ERD comprises the amino acid sequence of any one of SEQ ID NOs: 16-51 or an amino acid sequence having at least 70% sequence identity thereto.

9. A polynucleotide encoding a polypeptide, wherein the polypeptide comprises a tandem endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), wherein the tandem ERD comprises at least two ERDs.

10. The polynucleotide of claim 9, wherein the tandem ERD comprises a first ERD and a second ERD, wherein the first ERD comprises a first sequence derived from a first virus and the second ERD comprises a second sequence derived from a second virus.

11. The polynucleotide of claim 10, wherein the first and / or second virus is selected from the group consisting of: equine infectious anemia virus (EIAV), human immunodeficiency virus (HIV), Ebola (EBOV), Mason-Pfizer monkey virus (MPMV), Human T- lymphotropic virus (HTLV), vaccinia virus (Vacc.), tick-borne encephalitis virus (TBEV), simian virus (SV-5), rous sarcoma virus, porcine endogenous retrovirus (PERV), prototype foamy virus (PFV), and feline immunodeficiency virus (FIV).

12. The polynucleotide of claim 10 or 11, wherein the first virus and the second virus are the same virus.

13. The polynucleotide of claim 10 or 11, wherein the first virus and the second virus are not the same virus.

14. The polynucleotide of any one of claims 10 - 13, wherein the first and / or second sequence comprises an ESCRT-recruiting motif (ERM).

15. The polynucleotide of claim 14, wherein the ERM binds one or more ESCRT proteins.

16. The polynucleotide of claim 15, wherein the one or more ESCRT proteins is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4- like protein.

17. The polynucleotide of any one of claims 14-16, wherein the ERM comprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL, LXXLF, PPXY, LXXLXXL, L(X)nLXXLXXL, PYXE, and YXXL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5.

18. The polynucleotide of any one of claims 14-17, wherein the ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto.

19. The polynucleotide of claim 14, wherein the first sequence derived from a virus comprises a first ERM and the second sequence derived from a virus comprises a second ERM; wherein the first ERM is N-terminal to the second ERM; wherein the first ERM is PTAP, PSAP, PPxY, or YP(X)nL and the second ERM is LXXLF, LXXLXXL, LYPDLSEI, or IYPVRSNSTI; and wherein X is any amino acid and n is 1, 2, 3, 4, or 5.

20. The polynucleotide of any one of claims 14 - 19, wherein the first and / or the second ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto.

21. The polynucleotide of any one of claims 9-20, wherein the tandem ERD comprises a linker.

22. The polynucleotide of claim 21, wherein the linker is a flexible linker.

23. The polynucleotide of claim 21, wherein the linker is a Glycine-Serine linker.

24. The polynucleotide of claim 22 or 23, wherein the linker comprises the sequence of any one of SEQ ID NOs: 235-241.

25. The polynucleotide of any one of claims 9-24, wherein the tandem ERD comprises the amino acid sequence of any one of SEQ ID NOs: 52-116 or an amino acid sequence having at least 70% sequence identity thereto.

26. A polynucleotide encoding a polypeptide, wherein the polypeptide comprises a nonhuman homolog endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), wherein the non-human homolog ERD comprises a truncation relative to a naturally occurring non-human ERD.

27. The polynucleotide of claim 26, wherein the truncation relative to a naturally occurring non-human ERD comprises a truncation of 15 or less amino acids to the N terminus and / or the C terminus of a motif responsible for binding to one or more ESCRT proteins.

28. The polynucleotide of claim 26 or 27, wherein the naturally occurring non-human ERD is derived from a non-human vertebrate.

29. The polynucleotide of claim 28, wherein the non-human vertebrate is selected from the group consisting of: chicken, toad, mouse, elephant shrew, sunbittem, crocodile, Burmese python, fruit bat, pond turtle, and slow loris.

30. The polynucleotide of claim 26, wherein the naturally occurring ERD is derived from a fish.

31. The polynucleotide of claim 30, wherein the fish is selected from the group consisting of: zebrafish, greater pipefish, Atlantic halibut, snake pipefish, milkfish, European plaice, broad-nosed pipefish, banded pipefish, and goldfish.

32. The polynucleotide of any one of claims 26-31, wherein the non-human homolog ERD comprises an amino acid sequence of SEQ ID NOs: 117-136, or an amino acid sequence having at least 70% sequence identity thereto, or a portion thereof.

33. A polynucleotide encoding a polypeptide, wherein the polypeptide comprises a synthetic endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), wherein the synthetic ERD comprises at least two ESCRT-recruiting motifs (ERMs).

34. The polynucleotide of claim 33, wherein the at least two ERMs bind to one or more proteins associated with ESCRT.

35. The polynucleotide of claim 34, wherein the one or more proteins associated with ESCRT is tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), or a NEDD4-like protein.

36. The polynucleotide of any one of claims 33-35, wherein each ERM comprises a sequence selected from the group consisting of: P(T / S)AP, YP(X)nL, LXXLF, PPXY,LXXLXXL, L(X)nLXXLXXL, PYXE, and YXXL, wherein X is any amino acid and wherein n is 1, 2, 3, 4, or 5.

37. The polynucleotide of any one of claims 33-36, wherein each ERM comprises the sequence of any one of SEQ ID NOs: 4-15 and 354-364, or an amino acid sequence having at least 70% sequence identity thereto.

38. The polynucleotide of any one of claims 33-36, wherein the synthetic ERD further comprises a linker.

39. The polynucleotide of claim 38, wherein the synthetic ERD comprises, from N-terminus to C-terminus: a. a first ERM, a linker, and a second ERM; b. a linker, a first ERM, a linker, a second ERM, and a linker; c. a first ERM, a linker, a second ERM, a linker, and a third ERM; d. a linker, a first ERM, a linker, a second ERM, a linker, a third ERM, and a linker; e. a first ERM, a linker, a second ERM, a linker, a third ERM, a linker, and a fourthERM; or f. a linker, a first ERM, a linker, a second ERM, a linker, a third ERM, a linker, a fourth ERM, and a linker.

40. The polynucleotide of claim 38 or 39, wherein the linker is a flexible linker.

41. The polynucleotide of claim 40, wherein the linker is a Glycine-Serine (GS or SG) linker.

42. The polynucleotide of claim 41, wherein the GS linker comprises the sequence of any one of SEQ ID NOs: 235-241.

43. The polynucleotide of claim 38 or 39, wherein the linker is a rigid linker.

44. The polynucleotide of claim 43, wherein the rigid linker comprises the sequence of SEQID NO: 242 or SEQ ID NO: 243.

45. The polynucleotide of any one of claims 33-44, wherein the synthetic ERD comprises an ERM that binds to a NEDD4-like protein and an ERM that binds to ALIX, wherein the ERM that binds to a NEDD4-like protein is N-terminal to the ERM that binds to ALIX.

46. The polynucleotide of any one of claims 33-44, wherein the synthetic ERD comprises a first ERM comprising a sequence of PTAP, PSAP, PPXY, or YP(X)nL and a second ERM comprising a motif different from that of the first ERM, wherein said first ERM is N- terminal to said second ERM.

47. The polynucleotide of any one of claims 33-44, wherein the synthetic ERD comprises a first ERM comprising a sequence of LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI and a second ERM comprising a motif different from that of the first ERM, wherein said first ERM is C-terminal to said second ERM.

48. The polynucleotide of any one of claims 33-44, wherein the synthetic ERD comprises an ERM comprising a sequence of PTAP, PSAP, PPXY, or YP(X)nL and an ERM comprising a sequence of LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI, wherein said PTAP, PSAP, PPXY, or YP(X)nL sequence is N-terminal to said LXXLF, LXXLXXL, LYPDLSEI, PYKELYPL, or IYPVRSNSTI sequence.

49. The polynucleotide of any one of claims 33-48, wherein the synthetic ERD comprises the amino acid sequence of any one of SEQ ID NOs: 180-234 and 365-367, or an amino acid sequence having at least 70% sequence identity thereto.

50. A polynucleotide encoding a polypeptide, wherein the polypeptide comprises a synthetic coiled-coil endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), wherein the synthetic coiled-coil ERD comprises an amino acid sequence capable of binding one or more proteins associated with ESCRT; and wherein the synthetic coiled- coil ERD comprises a backbone capable of forming a coiled-coil structure.

51. The polynucleotide of claim 50, wherein the one or more proteins associated with ESCRT is selected from the group consisting of: tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (ALIX), and a NEDD4-like protein.

52. The polynucleotide of claim 51, wherein the one or more proteins associated with ESCRT is TSGlOl and / or ALIX.

53. The polynucleotide of any one of claims 50-52, wherein the synthetic coiled-coil ERD is computationally designed based on the structure of the ESCRT- and ALIX-binding region (EABR) sequence from the human CEP55 protein.

54. The polynucleotide of any one of claims 50-53, wherein the synthetic coiled-coil ERD has increased binding affinity to one or more proteins associated with ESCRT relative to the EABR sequence from the human CEP55 protein.

55. The polynucleotide of any one of claims 50-54, wherein the backbone comprises an amino acid sequence capable of forming an alpha helix.

56. The polynucleotide of claim 55, wherein the alpha helix interacts with another alpha helix formed by an identical sequence to make a 2-stranded coiled-coil structure.

57. The polynucleotide of any one of claims 50-56, wherein the synthetic coiled-coil ERD comprises the amino acid sequence of any one of SEQ ID NOs: 140-179, 301-309, and 313-353, or an amino acid sequence having at least 70% sequence identity thereto.

58. The polynucleotide of any one of claims 50-54, wherein the backbone comprises a first animo acid sequence capable of forming an alpha helix and a second amino acid sequence capable of forming an alpha helix.

59. The polynucleotide of claim 58, wherein the first animo acid sequence capable of forming an alpha helix and the second amino acid sequence capable of forming an alpha helix are linked by a linker.

60. The polynucleotide of claim 59, wherein the linker links the C-terminus of the first animo acid sequence to the N-terminus of the second amino acid sequence.

61. The polynucleotide of claim 59 or 60, wherein the linker is a flexible linker.

62. The polynucleotide of claim 59 or 60, wherein the linker is a rigid linker.

63. The polynucleotide of any one of claims 59 - 62, wherein the length of the linker is about 1 / 5 to 1 / 3 of the number of amino acids of the first or second amino acid sequence capable of forming an alpha helix.

64. The polynucleotide of any one of claims 58-63, wherein the synthetic coiled-coil ERD comprises the amino acid sequence of any one of SEQ ID NOs: 244-250 and 349-353, or an amino acid sequence having at least 70% sequence identity thereto.

65. The polynucleotide of any one of claims 50-56, wherein the backbone comprises an amino acid sequence derived from yeast GCN4.

66. The polynucleotide of claim 65, wherein the amino acid sequence derived from yeast GCN4 comprises the sequence of any one of SEQ ID NOs: 137-139 or an amino acid sequence having at least 70% sequence identity thereto.

67. The polynucleotide of any one of claims 50-56, wherein the synthetic coiled-coil ERD comprises a chimeric sequence, wherein the chimeric sequence comprises a portion of a first ERD capable of forming a coiled-coil structure fused in frame to a portion of a second ERD capable of forming a coiled-coil structure.

68. The polynucleotide of claim 67, wherein the first ERD and / or the second ERD is a nonhuman homolog ERD.

69. The polynucleotide of claim 68, wherein the non-human homolog ERD comprises the sequence of any one of SEQ ID NOs: 119-136 or an amino acid sequence having at least 70% sequence identity thereto.

70. The polynucleotide of claim 67, wherein the first ERD and / or the second ERD comprises the sequence of any one of SEQ ID NOs: 137-179, 252, 301-309, and 313-348, or an amino acid sequence having at least 70% sequence identity thereto.

71. The polynucleotide of claim 70, wherein the synthetic coiled-coil ERD comprises the amino acid sequence of SEQ ID NO: 348 or SEQ ID NO: 353 or an amino acid sequence having at least 70% sequence identity thereto.

72. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises:(a) an antigenic polypeptide; and(b) an endosomal sorting complex required for transport (ESCRT)-recruiting domain (ERD), wherein the ERD is:(i) a viral ERD;(ii) a tandem ERD;(iii) a non-human homolog ERD;(iv) a synthetic ERD comprising at least two ERMs; or(v) a synthetic coiled-coil ERD.

73. The polynucleotide of claim 72, wherein the antigenic polypeptide is derived from a protein of a virus.

74. The polynucleotide of claim 73, wherein the virus is hMPV, PIV3, respiratory syncytial virus, VZV, CMV, HSV1, HSV2, EBV, a coronavirus, influenza, a flavivirus, or orthopoxvirus.

75. The polynucleotide of claim 74, wherein the coronavirus is MERS-CoV, SARS-CoV, or SARS-CoV-2.

76. The polynucleotide of claim 75, wherein the protein is spike protein and the virus is MERS-CoV.

77. The polynucleotide of claim 75, wherein the protein is spike protein and the virus is SARS- CoV.

78. The polynucleotide of claim 75, wherein the protein is spike protein and the virus is SARS- CoV-2.

79. The polynucleotide of claim 74, wherein the protein is fusion (F) protein and the virus is hMPV.

80. The polynucleotide of claim 74, wherein the protein is fusion (F) protein and the virus is PIV3.

81. The polynucleotide of claim 74, wherein the protein is fusion (F) protein and the virus is respiratory syncytial virus.

82. The polynucleotide of claim 74, wherein the protein is glycoprotein E (gE) and the virus is VZV.

83. The polynucleotide of claim 74, wherein the protein is glycoprotein H (gH), glycoprotein L (gL), or glycoprotein B (gB) and the virus is CMV.

84. The polynucleotide of claim 74, wherein the protein is glycoprotein C (gC) or glycoprotein D (gD) and the virus is HSV1 or HSV2.

85. The polynucleotide of claim 72, wherein the antigenic polypeptide is derived from a protein of a bacterium.

86. The polynucleotide of claim 85, wherein the bacterium is an acne-causing bacterium, Staphylococcus, Borrelia, E. Coll, or Chlamydia.

87. The polynucleotide of claim 86, wherein the protein is DsAl and the acne-causing bacterium is Cutibacterium acnes.

88. The polynucleotide of claim 86, wherein the protein is esxA or esxB and the bacterium is Staphylococcus .

89. The polynucleotide of claim 86, wherein the protein is OspA and the bacterium is Borrelia, optionally Borrelia burgdorferi .

90. The polynucleotide of claim 86, wherein the protein is FimH and the bacterium is E. Coli.

91. The polynucleotide of claim 86, wherein the protein is major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), or OmcB and the bacterium is Chlamydia.

92. The polynucleotide of any one of claims 72-91, wherein the ERD is a viral ERD comprising a sequence derived from a virus.

93. The polynucleotide of any one of claims 72-91, wherein the ERD is a tandem ERD comprising at least two ERDs.

94. The polynucleotide of any one of claims 72-91, wherein the ERD is a non-human homolog ERD.

95. The polynucleotide of any one of claims 72-91, wherein the ERD is a synthetic ERD comprising at least two ESCRT-recruiting motifs (ERMs).

96. The polynucleotide of any one of claims 72-91, wherein the ERD is a synthetic coiled-coil ERD, wherein the synthetic coiled-coil ERD comprises an amino acid sequence capable of binding one or more proteins associated with ESCRT, wherein the synthetic ERD comprises a backbone capable of forming a coiled-coil structure.

97. The polynucleotide of any one of claims 1-96, wherein the polynucleotide comprises RNA.

98. The polynucleotide of claim 97, wherein the polynucleotide comprises mRNA.

99. The polynucleotide of any one of claims 1-96, wherein the polynucleotide comprises DNA.

100. A polypeptide encoded by the polynucleotide of any one of claims 1-99.

101. A cell expressing on its surface the fusion protein, or a portion of the fusion protein, encoded by the polynucleotide of any one of claims 72-99.

102. An enveloped virus-like particle (eVLP) comprising the fusion protein encoded by the polynucleotide of any one of claims 72-99.

103. An enveloped virus-like particle (eVLP) displaying on its surface the antigenic polypeptide, or part of the antigenic polypeptide, of the fusion protein encoded by the polynucleotide of any one of claims 72-99.

104. The eVLP of claim 102 or 103, wherein the diameter of the eVLP is 10 nm - 150 nm.

105. A vector comprising the polynucleotide of any one of claims 1- 99.

106. The vector of claim 105, wherein the vector is a viral vector.

107. The vector of claim 105, wherein the vector is a non-viral vector.

108. The vector of claim 107, wherein the non-viral vector is a plasmid.

109. The vector of claim 107, wherein the non-viral vector is a lipid nanoparticle (LNP).

110. The vector of claim 109, wherein the non-viral vector is a lipid nanoparticle (LNP) and the polynucleotide comprises mRNA.

111. A method of preventing or treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of any one of the polynucleotides of any one of claims 72-99, the eVLP of any one of claims 102-104, or the vector of any one of claims 105-110.

112. The method of claim 111, wherein the subject is a human subject.

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