Escrt-independent EVLP inducing domains

Abstract

Disclosed herein are polynucleotides that encode fusion proteins, wherein the fusion proteins comprise an antigenic polypeptide and an ESCRT-independent eVLP inducing domain (EIEIDo) and polypeptides encoded by the same. Also provided herein are related compositions, methods of making, and methods of using. In certain embodiments, EIEIDos and fusion proteins of the disclosure are useful for the production of enveloped virus-like particles (eVLPs), for use in the prevention and treatment of diseases or disorders.

Description

ESCRT-INDEPENDENT EVLP INDUCING DOMAINSRELATED APPLICATION

[0001] The present application claims the priority and benefits of U.S. Provisional Application No. 63 / 677,319, filed July 30, 2024. the contents of which are incorporated by reference herein in their entirety.STATEMENT CONCERNING GOVERNMENT SUPPORT

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

[0003] The contents of the electronic sequence listing (VCCN_012_01WO_SeqList_ST26.xml; Size: 250,230 bytes; and Date of Creation: July 30, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0004] Extracellular vesicles can be produced in a number of ways from host cells. The Endosomal Sorting Complex Required for Transport (ESCRT) is thought to be an important component of the 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. Other proteins, which have been previously shown to not require ESCRT (ESCRT-independent), have also been identified and enable transit and extrusion of proteins in extracellular vesicles in a similar manner.

[0005] Vaccine designs that incorporate human-derived ESCRT-recruiting domains (ERDs) may enhance antigenicity and / or immunogenicity by inducing antigen expression on cell surfaces and promoting the formation of extracellular vesicles presenting antigens, known as eVLPs. However, ERDs rely on ESCRT machinery, which may limit their use in some instances Thus, there is a need for ESCRT-independent methods that are reliant on different host cell machinery, or simply not reliant on host cell machinery, for producing eVLPs. Provided hereinare domains distinct from ERDs, and methods for generating and inducing eVLPs that are independent of host ESCRT machinery. Disclosed are methods and compositions that address the forgoing.SUMMARY

[0006] In one aspect, provided herein is a polynucleotide encoding a fusion protein, wherein the fusion protein comprises:(a) an antigenic polypeptide; and(b) a polypeptide comprising an ESCRT-independent eVLP inducing domain (EIEIDo).

[0007] In some embodiments of the polynucleotides of the disclosure, the EIEIDo comprises:(a) an envelope protein of a virus, or a domain or fragment thereof;(b) a tetraspanin polypeptide, or a domain or fragment thereof;(c) a binding partner of a tetraspanin polypeptide;(d) a Lysosome- Associated Membrane Protein 2 (LAMP2) polypeptide, or a domain or fragment thereof;(e) a binding partner of a LAMP2 polypeptide;(f) a phosphatidylserine-binding polypeptide, or a domain or fragment thereof;(g) a Platelet-Derived Growth Factor Receptor (PDGFR) polypeptide, or a domain or fragment thereof; or(h) a polypeptide comprising an amphipathic helix.

[0008] In some embodiments of the polynucleotides of the disclosure, the EIEIDo comprises an envelope protein of a virus, or a fragment thereof. In some embodiments, the EIEIDo comprises the cytoplasmic tail of the envelope protein. In some embodiments, the virus is a coronavirus. In certain embodiments, the coronavirus is a Gammacoronavirus, a Betacoronavirus, or an Alphacoronavirus. In some embodiments, the virus is selected from the group consisting of: Severe Acute Respiratory' Syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, avian infectious bronchitis virus (AIBV), mouse hepatitis virus (MHV), and transmissible gastroenteritis virus (TGEV). In some embodiments, the envelope protein comprises the amino acid sequence of any one of SEQ ID NOS: 6-9, or an ammo acid sequence having at least 70% sequence identity thereto.

[0009] In some embodiments of the polynucleotides of the disclosure, the EIEIDo comprises a tetraspanin polypeptide, or a domain or fragment thereof. In some embodiments, the tetraspaninpolypeptide comprises a human tetraspanin polypeptide or a homologous tetraspanin polypeptide from a non-human vertebrate species or an invertebrate species. In certain embodiments, the tetraspanin polypeptide comprises a human tetraspanin polypeptide. In certain embodiments, the tetraspanin polypeptide comprises a tetraspanin polypeptide from a nonhuman vertebrate species or an invertebrate species. In some embodiments, the non-human vertebrate species or invertebrate species is selected from the group consisting of: Spanish mole, brushtail possum, western clawed frog, Chinese alligator, common sole fish, Atlantic canary. Florida worm lizard, Adelie penguin, Common toad, Orbiculate cardinalfish, Aardvark, Diamondback terrapin, MacQueen's bustard, Green anole, Common wombat, Budorcas taxicolor, Patagioenas fasciata, Maylandia zebra. Hypanus sabinus, Echinops telfairi. Pan troglodytes, Lagopus leucura, Thamnophis elegans, Omithorhynchus anatinus, Etheostoma spectabile, Carassius Carassius, Pleuronectes platessa, Monodon monoceros, Motacilla alba alba, Syngnathus typhle, Xiphophorus couchianus, Lonchura striata, Meriones unguiculatus. Vicugna pacos, Podarcis muralis, Sinocyclocheilus anshuiensis, Sorex fumeus, Ursus arctos. Myotis davidii, Bos taurus, Gopherus flavomarginatus, Orycteropus afer afer, Phasianus colchicum, Actinia equina, Orbicella faveolata, Daphnia carinata, Microtus oregoni, Petaurus breviceps papuanus, Gallus gallus, Myotis lucifugus, Malaclemys terrapin pileata, Bufo bufo , Zonotrichia leucophrys gambelii, Anolis sagrei. Leopardus geoffroyi, Manacus candei. and Sapajus apella.

[0010] In some embodiments of the polynucleotides of the disclosure where the EIEIDo comprises a tetraspanin polypeptide, the tetraspanin polypeptide is derived from a protein selected from the group consisting of: CD9, CD63, CD81, CD82. CD37, CD53, CD151, CD231, tetraspanm (TSP)-l, TSP-2, TSP-3, TSP-4, TSP-5, TSP-6, TSP-9, TSP-11, and proteins encoded by genes TSPAN1-TSPAN33.

[0011] In some embodiments, the tetraspanin polypeptide is derived from CD9. In certain embodiments, the tetraspanin polypeptide is derived from human CD9 or CD9 from Aardvark, Diamondback terrapin, MacQueen's bustard, Green anole, Common wombat, Budorcas taxicolor, Patagioenas fasciata, Maylandia zebra, Hypanus sabinus, Echinops telfairi. Pan troglodytes, Lagopus leucura, Thamnophis elegans, Omithorhynchus anatinus, Etheostoma spectabile, Carassius Carassius, Pleuronectes platessa, Monodon monoceros, Motacilla alba alba, or Syngnathus typhle. In some embodiments, the tetraspanin polypeptide comprises theamino acid sequence of any one of SEQ ID NOS: 10, 138-152, and 223-227, or an amino acid sequence having at least 70% sequence identity thereto.

[0012] In some embodiments, the tetraspanin polypeptide is derived from CD63. In some embodiments, the tetraspanin polypeptide is derived from human CD63 or CD63 from Actinia equina. Orbicella faveolata, Daphnia carinata. Microtus oregoni, Petaurus breviceps papuanus. Gallus gallus. Myotis lucifugus, Malaclemys terrapin pileata, Bufo bufo, Zonotrichia leucophrys gambelii, Sorex fumeus, Anolis sagrei, Leopardus geoffroyi, Manacus candei, or Sapajus apella. In some embodiments, the tetraspanin polypeptide comprises a modification in an endosometargeting signal in CD63. In certain embodiments, the tetraspanin polypeptide comprises a modification of a tyrosine (Y) in the endosome-targeting signal in CD63. In certain embodiments, the modification is an amino acid substitution. In certain embodiments, the tetraspanin polypeptide comprises a modification of a glutamate (E) in the endosome-targeting signal in CD63. In certain embodiments, the tetraspanin polypeptide comprises a modification of a valine (V) in the endosome-targeting signal in CD63. In certain embodiments, the tetraspanin polypeptide comprises a modification of a methionine (M) in the endosome-targeting signal in CD63. In some embodiments, the tetraspanin polypeptide comprises an amino acid substitution of tyrosine (Y) to alanine (A) in the endosome-targeting signal in CD63. In certain embodiments, the tetraspanin polypeptide comprises a modification at a position corresponding to position 235, 236, 237, and / or 238 relative to SEQ ID NO: 1 1 . In certain embodiments, the tetraspanin polypeptide comprises a modification at a position corresponding to position 235 relative to SEQ ID NO: 11. In particular embodiments, the modification is an amino acid substitution of tyrosine (Y) to alanine (A).

[0013] In some embodiments, the tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 11 and 168-183, or an amino acid sequence having at least 70% sequence identity thereto.

[0014] In some embodiments, the tetraspanin polypeptide is derived from CD81. In some embodiments, the tetraspamn polypeptide is derived from human CD81 or CD81 from Atlantic canary, Florida worm lizard, Adelie penguin, Common toad, Orbiculate cardinalfish, Xiphophorus couchianus, Lonchura striata, Meriones unguiculatus, Vicugna pacos, Hypanus sabinus, Podarcis muralis, Sinocyclocheilus anshuiensis, Sorex fumeus, Ursus arctos, Myotis davidii, Patagioenas fasciata, Bos taurus, Gopherus flavomarginatus, Orycteropus afer afer, or Phasianus colchicus. In certain embodiments, the tetraspanin polypeptide comprises the aminoacid sequence of any one of SEQ ID NOS: 12, 153-167, and 218-222, or an amino acid sequence having at least 70% sequence identity thereto.

[0015] In some embodiments of the poly nucleotides of the disclosure, the EIEIDo comprises a chimeric tetraspanin polypeptide, where the chimeric tetraspanin polypeptide comprises a first tetraspanin polypeptide, and where a domain in that first tetraspamn polypeptide is substituted with a domain from a second tetraspanin polypeptide. In some embodiments, the first and second tetraspanin polypeptides are different. In some embodiments, the first and second tetraspanin polypeptides are independently selected from the group consisting of: CD9, CD63, CD81, CD82, CD37. CD53, CD151, CD231, tetraspanin (TSP)-l, TSP-2, TSP-3, TSP-4, TSP-5, TSP-6, TSP-9, TSP-11, proteins encoded by genes TSPAN1-TSPAN33, and domains or fragments thereof. In certain embodiments, the chimeric tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 184-205, or an amino acid sequence having at least 70% sequence identity thereto.

[0016] In some embodiments of the polynucleotides of the disclosure, the EIEIDo further comprises a PDZ1 domain. In certain embodiments, the EIEIDo comprising a PDZ1 domain comprises the amino acid sequence of SEQ ID NO: 206 or 207, or an amino acid sequence having at least 70% sequence identity thereto.

[0017] In some embodiments of the polynucleotides of the disclosure, the EIEIDo comprises a binding partner of a tetraspanin polypeptide, or a domain or fragment thereof. In some embodiments, the tetraspanin polypeptide is CD63. In some embodiments, the binding partner is a human cy tomegalovirus (HCMV) protein. In some embodiments, the HCMV protein is glycoprotein M (gM). In some embodiments, the binding partner comprises the cytoplasmic tail of HCMV gM. In certain embodiments, the cytoplasmic tail of HCMV gM comprises SEQ ID NO: 13 or an amino acid sequence having at least 70% sequence identity thereto.

[0018] In some embodiments of the poly nucleotides of the disclosure, the EIEIDo comprises a Lysosome- Associated Membrane Protein 2 (LAMP2) polypeptide, or a domain or fragment thereof. In some embodiments, the EIEIDo comprises a LAMP2A polypeptide, a LAMP2B polypeptide, or a LAMP2C polypeptide. In some embodiments, the LAMP2 polypeptide comprises a human LAMP2 polypeptide or a homologous LAMP2 polypeptide from a nonhuman vertebrate species or an invertebrate species. In certain embodiments, the LAMP2 polypeptide comprises a human LAMP2 polypeptide. In certain embodiments, the LAMP2 polypeptide comprises a LAMP2 polypeptide from a non-human vertebrate species or aninvertebrate species. In certain embodiments, the LAMP2 polypeptide comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence having at least 70% sequence identity thereto.

[0019] In some embodiments of the polynucleotides of the disclosure, the EIEIDo comprises a binding partner of a LAMP2 polypeptide, or a domain or fragment thereof. In some embodiments, the binding partner comprises a motif, where the motif is configured to help a LAMP2 polypeptide redirect proteins to eVLPs. In certain embodiments, the motif comprises the amino acid sequence KFERQ (SEQ ID NO: 17). In some embodiments, the binding partner comprises an ExoSignal, wherein the ExoSignal is a sequence that associates with a LAMP2 protein. In some embodiments, the binding partner comprises at least 2, at least 3, at least 4, or at least 5 copies of an ExoSignal. In some embodiments, the ExoSignal comprises the amino acid sequence VKKDQAEPLHRKFERQ (SEQ ID NO: 18).

[0020] In some embodiments of the polynucleotides of the disclosure where the EIEIDo comprises a binding partner of a LAMP2 polypeptide, the EIEIDo comprises the amino acid sequence of any one of SEQ ID NOS: 18-20, or an amino acid sequence having at least 70% sequence identity' thereto.

[0021] In some embodiments of the polynucleotides of the disclosure, the EIEIDo comprises a phosphatidyl serine-binding polypeptide, or a domain or fragment thereof. In some embodiments, the phosphatidylserine-binding polypeptide comprises a lactadherin polypeptide. In certain embodiments, the lactadherin polypeptide comprises a C1C2 domain. In some embodiments, the phosphatidylserine-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 70% sequence identity thereto.

[0022] In some embodiments of the polynucleotides of the disclosure, the EIEIDo comprises a Platelet-Derived Growth Factor Receptor (PDGFR) polypeptide, or a domain or fragment thereof. In some embodiments, the PDGFR polypeptide, or domain or fragment thereof, comprises a human PDGFR polypeptide or a homologous PDGFR polypeptide from a nonhuman vertebrate species or an invertebrate species. In some embodiments, the PDGFR polypeptide comprises a human PDGFR polypeptide. In some embodiments, the PDGFR polypeptide comprises a PDGFR polypeptide from a non-human vertebrate species or an invertebrate species. In certain embodiments, the homologous PDGFR polypeptide is selected from the group consisting of: Spanish mole, brushtail possum, western clawed frog, Chinese alligator, and the common sole fish.

[0023] In some embodiments, the PDGFR polypeptide, or domain or fragment thereof, comprises a transmembrane domain of a PDGFR polypeptide. In some embodiments, the PDGFR poly peptide comprises the amino acid sequence of any one of SEQ ID NOS: 22 and 228-232, or an amino acid sequence having at least 70% sequence identity thereto.

[0024] In some embodiments of the polynucleotides of the disclosure, the ElElDo comprises a polypeptide capable of forming an amphipathic helix. In some embodiments, the EIEIDo is capable of inducing membrane fission by increasing membrane curvature when expressed in a cell. In some embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from the group consisting of: M2 protein of influenza A. monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii, septum sitedetermining protein MinD from a bacterium (e.g., E. coli), peroxisomal membrane protein Peroxin 11 (Pexl Ip), phospholipid N-methyltransferase PmtA from Agrobacterium tumefaciens (AtPmtA), N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (MurG) from E. coli (EcMurG), EH domain-binding mitotic phosphoprotein (Epsin 1), amphiphysin from Drosophila, protein interacting with C kinase 1 (PICK1), ADP- ribosylation factor 1 (Arfl), diglucosyldiacylglycerol synthase (DGS) from Acholeplasma laidlawii. Endophilin Al, mammalian amphiphysin 2. Clathrin Assembly Lymphoid-Myeloid leukemia protein (CALM), or alpha-synuclein (a-synuclein). In some embodiments, the polypeptide capable of forming an amphipathic helix comprises the amino acid sequence of any one of SEQ ID NOS: 23-40, or an amino acid sequence having at least 70% sequence identity thereto.

[0025] In some embodiments of the polynucleotides of the disclosure, the EIEIDo comprises at least two polypeptides capable of forming an amphipathic helix. In some embodiments, the at least two polypeptides are the same. In some embodiments, the at least two polypeptides are different. In some embodiments, the at least two polypeptide capable of forming an amphipathic helix are isolated or derived from the group consisting of: M2 protein of influenza A, monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii, septum sitedetermining protein MinD from a bacterium (e g., E. coli), peroxisomal membrane protein Peroxin 11 (Pexl Ip), phospholipid N-methyltransferase PmtA from Agrobacterium tumefaciens (AtPmtA), N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (MurG) from E. coli (EcMurG), EH domain-binding mitotic phosphoprotein (Epsin 1), amphiphysin from Drosophila, protein interacting with C kinase 1 (PICK1), ADP-ribosylation factor 1 (Arfl), di glucosyldiacylglycerol synthase (DGS) from Acholeplasma laidlawii, Endophilin Al, mammalian amphiphysin 2, Clathrin Assembly Lymphoid-Myeloid leukemia protein (CALM), or alpha-synuclein (a-synuclein). In some embodiments, the at least two polypeptides are linked in-frame. In some embodiments, the at least two polypeptides are linked by a linker. In some embodiments, the linker is a glycine serine linker. In certain embodiments, the linker comprises the sequence of any one of SEQ ID NOS: 209-217. In some embodiments, the EIEIDo comprises the amino acid sequence of any one of SEQ ID NOS: 41- 120, or an amino acid sequence having at least 70% sequence identity thereto.

[0026] In some embodiments of the polynucleotides of the disclosure, the fusion protein further comprises an endocytosis prevention motif (EPM).

[0027] In some embodiments of the polynucleotides of the disclosure, the antigenic polypeptide is derived from a viral protein. In some embodiments, the viral protein is derived from 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), a coronavirus, influenza, a flavivirus, or orthopoxvirus.

[0028] The polynucleotide of claim 84, wherein the viral protein is derived from a coronavirus, wherein the coronavirus is Middle East respiratory syndrome coronavirus (MERS-CoV). Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), or SARS-CoV-2.

[0029] In certain embodiments, the protein is spike protein. In certain embodiments, the viral protein is fusion (F) protein derived from hMPV, PIV3, or respiratory syncytial virus. In certain embodiments, the viral protein is:(a) glycoprotein E (gE) derived from N N,(b) glycoprotein H (gH), glycoprotein L (gL), or glycoprotein B (gB) derived from CMV; or(c) glycoprotein C (gC) or glycoprotein D (gD) derived from HSV1 or HSV2.

[0030] In some embodiments of the polynucleotides of the disclosure, the antigenic polypeptide is derived from a bacterial protein. In some embodiments, the bacterial protein is derived from an acne-causing bacterium. Staphylococcus, Borrelia, E. Coli, or Chlamydia. In certain embodiments, the bacterial protein is:(a) DsAl derived from Cutibacterium acnes;(b) esxA or esxB derived from Staphylococcus;(c) OspA derived from Borrelia, optionally Borrelia burgdorferi;(d) FimH derived from E. Coli; or(e) major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), or OmcB derived from Chlamy dia.

[0031] In some embodiments of the polynucleotides of the disclosure, the antigenic polypeptide is derived from a parasite associated protein.

[0032] In some embodiments of the polynucleotides of the disclosure, the antigenic polypeptide is derived from a cancer associated protein. In certain embodiments, the antigenic polypeptide is derived from a prostate cancer associated protein. In particular embodiments, the prostate cancer associated protein is Six Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) or Prostate-specific membrane antigen (PSMA). In certain embodiments, the antigenic polypeptide is derived from a melanoma associated protein. In particular embodiments, the melanoma associated protein is Tyrosinase or transmembrane phosphatase with tensin homology protein (TPTE).

[0033] In some embodiments of the polynucleotides of the disclosure, the polynucleotide comprises RNA.

[0034] In some embodiments of the polynucleotides of the disclosure, the polynucleotide comprises mRNA.

[0035] In some embodiments of the polynucleotides of the disclosure, the polynucleotide comprises DNA.

[0036] In some embodiments of the polynucleotides of the disclosure, the polynucleotide, or the EIEIDo or fusion protein encoded by the polynucleotide: a) allows for multivalent display of an antigenic polypeptide on an eVLP; b) allows for tailoring a lipid composition of eVLPs formed by cells expressing the fusion protein; c) enables altered modulation of eVLP surface presentation; d) enables incorporation of molecules, in addition to the antigenic polypeptide or fusion protein, into eVLPs formed by cells expressing the fusion protein; e) allows for controlling of the ratio of eVLP-associated and cell surface-associated antigens; and / or f) enables simultaneous production of a population of orthogonal eVLPs.

[0037] In another aspect, provided herein is an EIEIDo comprising:(a) an envelope protein of a virus, or a domain or fragment thereof;(b) a tetraspanin polypeptide, or a domain or fragment thereof;(c) a binding partner of a tetraspanin polypeptide;(d) a Lysosome- Associated Membrane Protein 2 (LAMP2) poly peptide, or a domain or fragment thereof;(e) a binding partner of a LAMP2 polypeptide;(f) a phosphatidylserine-binding polypeptide, or a domain or fragment thereof;(g) a Platelet-Derived Grow th Factor Receptor (PDGFR) polypeptide, or a domain or fragment thereof; or(h) a polypeptide comprising an amphipathic helix; where (a) to (h) does not comprise a naturally occurring sequence.

[0038] In another aspect, provided herein is a polypeptide encoded by the polynucleotide of the disclosure.

[0039] In another aspect, provided herein is a fusion protein encoded by polynucleotide of the disclosure.

[0040] In another aspect, provided herein is a fusion protein comprising any one of the sequences of SEQ ID NOS: 1-207 and 218-234, or a sequence having at least 70% sequence identity thereto.

[0041] In another aspect, provided herein is a cell expressing on its surface the fusion protein, or a portion of the fusion protein, of the disclosure.

[0042] In another aspect, provided herein is a cell expressing on its surface the antigenic polypeptide of the fusion protein, or a portion of the antigenic polypeptide of the fusion protein, of the disclosure.

[0043] In another aspect, provided herein is an enveloped virus-like particle (eVLP) comprising a polypeptide encoded by the polynucleotide of the disclosure or the fusion protein of the disclosure.

[0044] In another aspect, provided herein is an enveloped virus-like particle (eVLP) displaying on its surface the antigenic polypeptide, or a portion of the antigenic polypeptide, of the fusion protein of the disclosure. In some embodiments, the diameter of the eVLP ranges from about 10 nm to about 1000 nm.

[0045] In another aspect, provided herein is a vector comprising the polynucleotide of any one 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 certainembodiments, the non-viral vector comprises a lipid nanoparticle (LNP). In certain embodiments, the non-viral vector comprises a lipid nanoparticle (LNP) and the polynucleotide comprises mRNA.

[0046] In another aspect, provided 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, a polypeptide of the disclosure, a fusion protein of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vector of the disclosure.

[0047] In another aspect, provided herein is a method of vaccinating a subject in need thereof, comprising administering to the subject an effective amount of a polynucleotide of the disclosure, a polypeptide of the disclosure, a fusion protein of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vector of the disclosure.

[0048] In some embodiments of the methods of the disclosure, the subject has been diagnosed with an infection or is at risk of being infected by a pathogen.

[0049] In some embodiments of the methods of the disclosure, the subject has been diagnosed with an infection with a virus, a bacterium, or a parasite, or is at risk of being infected by a virus, a bacterium, or a parasite.

[0050] In some embodiments of the methods of the disclosure, the subject has been diagnosed w ith cancer or is at risk of cancer.

[0051] In some embodiments of the methods of the disclosure, the subject is a mammalian subject. In certain embodiments, the subject is a human subject.

[0052] In another aspect, provided herein is a poly nucleotide of the disclosure, a polypeptide of the disclosure, a fusion protein of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vector of the disclosure for use in the treatment or prevention of a disease or disorder.

[0053] In another aspect, provided herein is a poly nucleotide of the disclosure, a polypeptide of the disclosure, a fusion protein of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vector of the disclosure for use in vaccination against a disease or disorder.

[0054] In some embodiments of the polynucleotide of the disclosure, the polypeptide of the disclosure, the fusion protein of the disclosure, the cell of the disclosure, the eVLP of the disclosure, or the vector of the disclosure for use as described herein, the disease or disorder is an infection or a cancer. In certain embodiments, the infection is a viral infection, a bacterialinfection, or a parasitic infection. In certain embodiments, the cancer is prostate cancer or melanoma.

[0055] In another aspect, provided herein is a use a polynucleotide of the disclosure, a polypeptide of the disclosure, a fusion protein of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vector of the disclosure for manufacture of a medicament for the prevention or treatment of a disease or disorder.

[0056] In another aspect, provided herein is a use a polynucleotide of the disclosure, a polypeptide of the disclosure, a fusion protein of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vector of the disclosure for manufacture of a medicament for vaccination against a disease or disorder.

[0057] In some embodiments of the uses of the disclosure, the disease or disorder is an infection or a cancer. In certain embodiments, the infection is a viral infection, a bacterial infection, or a parasitic infection. In certain embodiments, the cancer is prostate cancer or melanoma.BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1. depicts a schematic of cells producing eVLPs in ESCRT-independent (left) or ESCRT-mediated (right) ways, in which immunogenic eLVPs are produced.

[0059] FIGS. 2A-2B depict an ESCRT-independent method for an antigenic polypeptide to be displayed on eVLPs by fusing the antigenic polypeptide to a tetraspanin protein or portion thereof (left) or by fusing the antigenic polypeptide to a viral peptide known to associate with tetraspanin proteins (right).

[0060] FIGS. 3A-3B depict an ESCRT-independent method for an antigenic polypeptide to be displayed on eVLPs by fusing the antigenic polypeptide to Lamp2a or Lamp2b proteins (left) or fusing the antigenic polypeptide to a polypeptide known to associate w ith Lamp2a / b proteins (termed exosignal, right).

[0061] FIG. 4 depicts an ESCRT-independent method for an antigenic polypeptide to be displayed on eVLPs by fusing the antigenic polypeptide to multiple copies of a polypeptide known to associate with Lamp2a / b proteins presented in tandem (termed exosignal).

[0062] FIG. 5 depicts an ESCRT-independent method for an antigenic polypeptide to be displayed on eVLPs by fusing the antigenic polypeptide to the C1C2 domain of the lactadherin protein.

[0063] FIG. 6 depicts an ESCRT-independent method for an antigenic polypeptide to be displayed on eVLPs by fusing the antigenic polypeptide to a Platelet-derived Growth Factor Receptor (PDGFR) transmembrane domain.

[0064] FIG. 7A depicts an ESCRT-independent method for an antigenic polypeptide to be displayed on eVLPs. by fusing the antigenic polypeptide to an amphipathic helix that can promote membrane budding / fission.

[0065] FIGS. 7B-7C depict a side view (top) and front view (bottom) of eVLPs comprising the amphipathic helix driving membrane curvature of the eVLPs.

[0066] FIGS. 8A-8D depict linear schematics depicting the possible insertion sites of ESCRT- independent enveloped virus-like particle inducing domains (EIEIDos) in a construct comprising an immunogenic peptide (e.g., an exemplary' MERS antigen as used in aspects of this disclosure).

[0067] FIG. 9 provides a dot blot quantifying eVLP levels in supernatant from HEK293T cells transfected with exemplary' constructs comprising MERS spike protein and EIEIDo sequences as detailed herein, via staining with MERS spike protein antibody D12.

[0068] FIG. 10 depicts a plot reflecting dot blot density' on the y-axis (derived from FIG. 9 and its replicates) to the total cell expression, as determined by a cell ELISA of fixed and permeabilized cells, on the x-axis. Also shown are inset plots of specific regions of the complete plot to highlight constructs with specific EIEDo sequences. Different shapes represent different ESCRT-independent mechanisms for eVLP induction (solid squares show' ESCRT-dependent sequences).

[0069] FIG. 11 depicts the StrepTactin® purification of eVLPs containing N-terminally strep- tagged MERS spike protein. Lead candidates as selected from FIG. 9 w ere expressed in a larger volume in Expi293F cells and purified via a StrepTactin® resin as depicted and further analyzed in subsequent figures.

[0070] FIGS. 12A-12B provide a western blot (FIG. 12A) and an SDS-Page gel (FIG. 12B) of isolated eVLPs. as selected from FIG. 9 and purified via StrepTactin®.

[0071] FIG. 13 provides a graph of size measurements of purified eVLPs, determined via dynamic light scattering, a tool to measure size of particles.

[0072] FIGS. 14A-14F provide transmission electron microscopy (TEM) images, including representative frames, of purified eVLPs induced by Lactadherin C1C2 domain (FIG. 14A). ExoSignal (FIG. 14B), EcMurG amphipathic helix (FIG. 14C), Drosophila amphiphysinamphipathic helix (FIG. 14D), CD81 (FIG. 14E), or human PDGFR transmembrane domain (FIG. 14F) fused to MERS spike protein.

[0073] FIGS. 15A-15C depict an ESCRT-independent method for an antigenic polypeptide to be displayed on eVLPs, by fusing the antigenic polypeptide to multiple amphipathic helices using a linker, that can promote membrane budding / fission. FIGS. 15B-15C depict a schematic illustrating a side view (top) or front view (bottom) of eVLPs comprising multiple amphipathic helices driving membrane curvature of the eVLPs. FIG. 15D depicts an ESCRT-independent method for an antigenic polypeptide to be displayed on eVLPs, by fusing the antigenic polypeptide to multiple amphipathic helices fused in frame, that can promote membrane budding / fission. FIGS. 15E-15F depict a side view (top) or front view (bottom) of eVLPs comprising multiple amphipathic helices driving membrane curvature of the eVLPs.

[0074] FIGS. 16A-16C provide graphs showing eVLP inducing activity' on the y-axis, as measured by dot blot, and percent sequence homology’ to corresponding human sequence on the x-axis of homologs of a PDGFR transmembrane domain (FIG. 16A), CD81 (FIG. 16B), and CD9 (FIG. 16C) from non-human species. The PDGFR transmembrane domain, CD81, and CD9 homologs were fused to MERS spike protein.

[0075] FIGS. 17A-17C provide exemplary schematics demonstrating a process for designing extended, “in frame” amphipathic helices.

[0076] FIGS. 17D-17G provide linear schematics depicting backbones used and the possible insertion sites of EIEIDos in constructs comprising West Nile Virus (WNV) NS1 monomer and homodimer as exemplary' antigenic peptides.

[0077] FIG. 18 provides a dot blot measuring membrane-bound NS 1 protein, detected using WNV NSl-specific antibody 22NS1, in cell supernatant from HEK293T cells transfected with constructs encoding WNV NS1 monomer (WNV mNSl) or homodimer (WNV dNSl) fused to EIEIDos, as detailed in Table 10.

[0078] FIG. 19 provides a dot blot measuring membrane-bound NS1 protein in cell supernatant from HEK293T cells transfected with constructs encoding WNV NS1 monomer (WNV mNSl) or homodimer (WNV dNSl) fused to EIEIDos and various transmembrane domains, as detailed in Table 11.

[0079] FIG. 20 provides a schematic of different tetraspanin-based construct designs intended to improve B and T cell responses against antigens.

[0080] FIGS. 21A-21B provide linear schematics depicting backbones used and the possible insertion sites of EIEIDos in constructs comprising signal peptide (Sig Pep), StrepTag II (Strep), WNV NS1 protein fused to Japanese Encephalitis Virus (JEV) NS1 by a SGSG linker (L), the transmembrane domain (TM) of Epstein-Barr Virus gp220 protein, and tetraspanin-based EIEIDos. Some constructs (FIG. 2 IB) also had an endocytosis prevention motif (EPM).

[0081] FIGS. 22A-22B provide dot blots measuring levels of WNV and JEV NS1 heterodimer, detected by flavi virus NS1 cross-reactive antibody 1G5.3 (FIG. 22A) or WNV NS1 specific antibody 22NS1 (FIG. 22B), in cell supernatant from HEK293T cells transfected with constructs based on tetraspanin designs as detailed in Table 12.

[0082] FIGS. 23A-23C provide dot blots (FIG. 23A and FIG. 23B) and absorbance at 450nm measured by ELISA (FIG. 23C) showing levels of WNV and JEV NS1 heterodimer, detected by anti-WNV NS1 antibody 22NS1 (FIG. 23A and FIG. 23C) or anti-JEV NS1 antibody JA12 (FIG. 23B) in cell supernatant from Expi293F cells transfected with various constructs as detailed in Table 13.

[0083] FIG. 24 provides a dot blot measuring levels of HLA class I co-packaged with tetraspanin-driven eVLPs, detected by mouse anti-human HLA- ABC antibody, in cell supernatant from Expi293F cells transfected with various constructs as detailed in Table 13.

[0084] FIG. 25 provides a graph showing serum antibody response, as measured by ELISA where MERS spike was directly coated to ELISA plates, in sera collected on day 35 from mice immunized with mRNA vaccines encoding MERS spike protein fused to different classes of EIEIDos.DETAILED DESCRIPTION

[0085] Disclosed herein are endosomal sorting complex required for transport (ESCRT)- independent enveloped virus-like particle (eVLP) inducing domains (EIEIDos), polypeptides comprising an EIEIDo, and polynucleotides encoding the same. Also disclosed herein are fusion proteins comprising an antigenic polypeptide and an EIEIDo, and polynucleotides encoding the same. In some embodiments, the EIEIDo is fused to the C-terminus of the antigenic polypeptide. In some embodiments, the EIEIDo is fused to the cytoplasmic end of the antigenic polypeptide.

[0086] Also disclosed herein are methods for producing eVLPs using an EIEIDo, a polypeptide, a fusion protein, and / or a polynucleotide of the disclosure. Additionally, disclosedherein are ESCRT-independent methods of producing eVLPs in the context of deliver}' of genetic material (e.g., DNA or RNA) in a vaccine setting.

[0087] Without being held to theory or mechanism, virus-like particles (VLPs) are non- infectious particles whose production is driven by a viral capsid protein; whereas the eVLPs of the disclosure are non-infectious membraned particles whose production does not require a viral capsid protein and whose production is instead driven by one or more polynucleotides, polypeptides, or fusion protein of the disclosure in a manner not dependent on host ESCRT machinery. Antigenic polypeptides, or portions thereof, are displayed on the surface of eVLPs when the antigenic polypeptides are fused to an EIEIDo.

[0088] As used herein, polypeptides independent of host ESCRT machinery may include polypeptides that are entirely independent of host ESCRT machinery, as well as polypeptides that interact with ESCRT machinery but whose mechanism for facilitating production of eVLPs is not entirely reliant upon ESCRT machinery to function. For example, ESCRT independence can in some cases be determined or defined as the ability of a domain to retain significant function in inducing and / or facilitating eVLP formation when traditional ESCRT pathways, or a component thereof, are inhibited. VPS4 is one non-limiting example of a protein crucial for ESCRT function. Thus, a non-limiting example of a context in which traditional ESCRT pathways in a cell are inhibited is overexpression of dominantly inhibitory VPS4 mutants which are capable of inhibiting traditional ESCRT pathways. Therefore, a domain capable of inducing and / or facilitating eVLP formation in a cell when a dominantly inhibitory' VPS4 mutant is expressed in the cell can be determined to be ESCRT independent.

[0089] In some embodiments, an EIEIDo of the disclosure comprises: (1) an envelope protein of a virus, or a domain or fragment thereof; (2) a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof; (3) a lysosome-associated membrane protein 2 (LAMP2) polypeptide, or a domain or fragment thereof, or a binding partner thereof; (4) a polypeptide that binds to phosphatidylserine, or a domain or fragment thereof; (5) a platelet- derived growth factor receptor (PDGFR) polypeptide, or a domain or fragment thereof; or (6) a polypeptide capable of forming an amphipathic helix.

[0090] Also provided herein are compositions, methods of making, and methods of using an EIEIDo, a polypeptide, a fusion protein, a polynucleotide, or an eVLP of the disclosure. In some embodiments, an EIEIDo, a polypeptide, a fusion protein, a polynucleotide of the disclosure is useful for producing eVLPs for use in the prevention and / or treatment of a disease or disorder. Insome embodiments, generation of eVLPs through ESCRT-independent mechanisms provides at least one advantage. For example, an EIEIDo, a polypeptide, a fusion protein, or a polynucleotide of the disclosure may be configured (1) to allow for multivalent display of an antigen on an eVLP, (2) to allow for tailoring a lipid composition of the resultant eVLPs, (3) to enable altered modulation of eVLP surface presentation, (4) to enable incorporation of other molecules (e.g., MHC molecules), in addition to the antigenic polypeptide or fusion protein, into the produced eVLPs, (5) to allow for controlling of the ratio of eVLP-associated and cell surface-associated antigens, and / or (6) to enable simultaneous, orthogonal eVLP production. In some embodiments, altered modulation of eVLP surface presentation comprises tuning of protein density and / or protein composition on the eVLP. Without being held to theory or mechanism, in some embodiments, an EIEIDo, a polypeptide, a fusion protein, or a polynucleotide of the disclosure configured as described herein can impact immune cell recognition. In some embodiments, ESCRT-independent mechanisms provide versatility, allowing for delivery of different types of proteins (e.g., transmembrane proteins, secreted proteins) on eVLPs. In some embodiments, the EIEIDos, polypeptides, fusion proteins, eVLPs, polynucleotides, and related compositions described herein are useful for producing vaccines and / or can be used as vaccines.Terms and Concepts

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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 nonnatural 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.

[0096] 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 companson are well-known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology' alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection. Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.

[0097] Depending on the algorithm, the calculated percent sequence identity may differ. For example, there are at least three ways in which to calculate a percent sequence identity.% Query sequence identity : = (Number of alignment identities) / (Length of Query' sequence); % Subject sequence identity: = (Number of alignment identities) / (Length of Subject sequence);%Alignment sequence identity' = (Number of alignment identities) / (Length of Alignment)

[0098] Accordingly, when the term “sequence identity ” is used herein, it can include any of the above non-limiting methodologies provided above to calculate.

[0099] Numeric ranges are inclusive of the numbers defining the range. Where a range of values is stated, each intervening integer value, and each fraction thereof, between the recitedupper and lower limits of that range is also specifically disclosed, as is each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0100] Where a value is explicitly stated, it is to be understood that values which are about the same quantity or amount as the stated value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0101] The term "antigenic” as used herein 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 bind to the products of an immune response, such as antibodies or T cell receptors. The antigenic polypeptides of the disclosure, in most cases, are also immunogenic, meaning they are capable of eliciting an immune response to an antigen in a subject. Various aspects of an immune response elicited by compositions comprising polynucleotides, antigenic polypeptides, fusion proteins, vectors, or eVLPs of the disclosure can be determined using standard assays, some of which are described in the present disclosure.ESCRT-independent enveloped virus-like particle (eVLP) inducing domains

[0102] Provided herein are endosomal sorting complex required for transport (ESCRT)- independent enveloped virus-like particle (eVLP) inducing domains (EIEIDos) which facilitate production of eVLPs. In some embodiments, EIEIDos of the disclosure comprise: (1) an envelope protein of a virus, or a domain or fragment thereof; (2) a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof; (3) a lysosome-associated membraneprotein 2 (LAMP2) polypeptide, or a domain or fragment thereof, or a binding partner thereof; (4) a a polypeptide that binds to phosphatidylserine, or a domain or fragment thereof; (5) a platelet-derived grow th factor receptor (PDGFR) poly peptide, or a domain or fragment thereof; and / or (6) a polypeptide capable of forming an amphipathic helix. In some embodiments, without being held to theory or mechanism. EIEIDos of the disclosure are configured to facilitate production of eVLPs.Viral Envelope Proteins

[0103] In some embodiments, an ESCRT-independent enveloped virus-like particle (eVLP) inducing domain (EIEIDo) of the disclosure comprises an envelope protein of a virus, or a domain or fragment thereof. As used herein, the term “envelope protein” is all encompassing, and inclusive of naturally occurring envelope proteins of a vims and engineered variants thereof. In certain embodiments, the vims is a coronavirus. Examples of coronavimses include, but are not limited to, a Gammacoronavims, a Betacoronavirus, an Alphacoronavirus, an avian infectious bronchitis virus (AIBV), a Mouse Hepatitis Virus (MHV), and transmissible gastroenteritis virus (TGEV). In some embodiments, an EIEIDo of the disclosure comprises an envelope protein of a betacoronavirus. Non-limiting examples of betacoronaviruses include Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. In certain embodiments, an EIEIDo of the disclosure comprises an envelope protein of SARS-CoV. In certain embodiments, an EIEIDo of the disclosure comprises an envelope protein of MERS-CoV. In certain embodiments, an EIEIDo of the disclosure comprises an envelope protein of SARS-CoV-2. In certain embodiments, an EIEIDo of the disclosure comprises an envelope protein of AIBV. In certain embodiments, an EIEIDo of the disclosure comprises an envelope protein of MHV. In certain embodiments, an EIEIDo of the disclosure comprises an envelope protein of TGEV.

[0104] In some embodiments, an EIEIDo of the disclosure comprises an entire envelope protein or a portion of an envelope protein, including the cytoplasmic tail, one or more domains, or one or more fragments. In some embodiments, without being held to theory or mechanism, an entire envelope protein or a portion thereof contributes to eVLP production, as depicted in FIG. 1 and exemplified in Example 1. In certain embodiments, a portion of an envelope protein contributes to eVLP production. In specific embodiments, the portion of an envelope protein comprises the cytoplasmic tail, one or more domains, and / or one or more fragments.

[0105] In some embodiments, an EIEIDo described herein comprises an envelope protein of a coronavirus, or a domain or fragment thereof. Table 1 provides exemplary sequences for envelope proteins of coronaviruses. In certain embodiments, the envelope protein comprises an amino acid sequence selected from Table 1. or an amino acid sequence having at least about 70%. at least about 71%. at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%. at least about 86%, at least about 87%, at least about 88%, at least about 89%. at least about 90%. at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In certain embodiments, the envelope protein comprises the amino acid sequence of any one of SEQ ID NOs: 6-9, or an amino acid sequence having at least about 70%, at least about 71%. at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%. at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0106] In some embodiments, an EIEIDo described herein comprises a portion of an envelope protein of a coronavirus. In certain embodiments, the portion comprises the cytoplasmic tail. In some embodiments, the cytoplasmic tail from an envelope protein of a coronavirus comprises the amino acid sequence of any one of SEQ ID NOs: 6-9, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%. at least about 81%, at least about 82%, at least about 83%, at least about 84%. at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.Table 1: Exemplary Cytoplasmic Tail Coronavirus Envelope Protein Amino Acid Sequences

[0107] In some embodiments, an EIEIDo described herein comprises an envelope protein of a coronavirus, wherein the envelope protein comprises an amino acid sequence selected from Table 1, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%. at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0108] Also disclosed herein are polypeptides comprising an EIEIDo described herein comprising an envelope protein of a coronavirus. It can be appreciated that any EIEIDo described herein comprising an envelope protein of a coronavirus may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, which are further discussed herein in detail. Provided herein are polypeptides comprising a sequence in Table 1.

[0109] Additionally, disclosed herein are polynucleotides encoding the polypeptides comprising an EIEIDo described herein comprising an envelope protein of a coronavirus and polynucleotides encoding fusion proteins comprising an EIEIDo described herein comprising an envelope protein of a coronavirus. In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprises an antigenic polypeptide described herein and an EIEIDo described herein comprising an envelope protein of a coronavirus.Provided herein are polynucleotides that encode any one or more of the sequences of Table 1, or a sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%. at least about 82%, at least about 83%, at least about 84%. at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0110] Polynucleotides can be DNA or RNA (e.g., mRNA), as described in detail herein.Tetraspanin Polypeptides[OHl] In some embodiments, an ESCRT-independent enveloped virus-like particle (eVLP) inducing domain (EIEIDo) of the disclosure comprises a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof. As used herein, the term “tetraspanin polypeptide” is all encompassing, and inclusive of naturally occurring tetraspanin polypeptides and engineered variants thereof. In some embodiments, when expressed with an antigenic polypeptide, the tetraspanin polypeptide facilitates generation of an eVLP with the antigenic polypeptide extending therefrom, as depicted in FIGS. 2A-2B and exemplified in Examples 1- 4.

[0112] In some embodiments, the EIEIDo comprises a tetraspanin polypeptide selected from the group consisting of: CD9, CD63, CD81, CD82, CD37, CD53, CD151, CD231, tetraspanin (TSP)-l, TSP-2, TSP-3, TSP-4, TSP-5, TSP-6, TSP-9, TSP-11, proteins encoded by genes TSPAN1-TSPAN33, and domains or fragments thereof. In some embodiments, the tetraspanin polypeptide comprises CD9, CD63, CD81, and / or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises CD9 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises CD63 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises CD81 or a domain or fragment thereof. In some embodiments, the tetraspanin polypeptide comprises CD82, CD37, CD53, CD151, CD231, TSP-2, TSP-4, TSP-5, TSP-9, TSP-11, and / or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises CD82 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises CD37 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises CD53 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptidecomprises CD151 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises CD231 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises TSP-2 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises TSP-4 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises TSP-5 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises TSP-9 or a domain or fragment thereof. In certain embodiments, the tetraspanin polypeptide comprises TSP-11 or a domain or fragment thereof.

[0113] Homologous tetraspanin polypeptides from any species may be utilized. For example, in some embodiments, homologous tetraspanin polypeptides from vertebrates or invertebrates are used. Examples of vertebrates or invertebrates from which tetraspanin polypeptides can be derived include, but are not limited to, Atlantic canary, Florida worm lizard, Adelie penguin, Common toad, Orbiculate cardinalfish, Aardvark, Diamondback terrapin, MacQueen's bustard, Green anole, and Common wombat. In some embodiments, a tetraspanin polypeptide is derived from a mammal. In some embodiments, a tetraspanin polypeptide is derived from a vertebrate. In some embodiments, a tetraspanin polypeptide is derived from a fish, a reptile, or a bird. In some embodiments, a tetraspanin polypeptide is derived from an invertebrate. In some embodiments, a tetraspanin polypeptide is derived from a species selected from the group consisting of Spanish mole, brushtail possum, western clawed frog, Chinese alligator, common sole fish, Atlantic canary', Florida worm lizard, Adelie penguin, Common toad, Orbiculate cardinalfish, Aardvark, Diamondback terrapin, MacQueen's bustard, Green anole, Common wombat, Budorcas taxicolor, Patagioenas fasciata, Maylandia zebra. Hypanus sabinus, Echinops telfairi, Pan troglodytes, Lagopus leucura, Thamnophis elegans. Ornithorhynchus anatinus, Etheostoma spectabile, Carassius Carassius, Pleuronectes platessa, Monodon monoceros, Motacilla alba alba, Syngnathus typhle, Xiphophorus couchianus, Lonchura striata, Meriones unguiculatus, Vicugna pacos, Podarcis muralis, Sinocyclocheilus anshuiensis, Sorex fumeus. Ur sus arctos, Myotis davidii, Bos taurus, Gopherus flavomarginatus, Orycteropus afer afer, Phasianus colchicum. Actinia equina, Orbicella faveolata. Daphnia carinata, Microtus oregoni, Petaurus breviceps papuanus, Gallus gallus, Myotis lucifugus, Malaclemys terrapin pileata, Bufo bufo , Zonotrichia leucophrys gambelii, Anolis sagrei, Leopardus geoffroyi, Manacus candei. and Sapajus apella.

[0114] In some embodiments, the tetraspanin polypeptide is derived from human CD9 or CD9 from Budorcas taxicolor, Patagioenas fasciata, Maylandia zebra, Hypanus sabinus, Echinops telfairi, Pan troglodytes, Lagopus leucura, Thamnophis elegans, Ornithorhynchus anatinus, Etheostoma spectabile, Carassius Carassius, Pleuronectes platessa, Monodon monoceros, Motacilla alba alba, Syngnathus typhle. Aardvark, Diamondback terrapin, MacQueen's bustard. Green anole, or Common wombat.

[0115] In some embodiments, the tetraspanin polypeptide is derived from human CD63 or CD63 from Actinia equina, Orbicella faveolata. Daphnia car inala. Microtus oregoni, Petaurus breviceps papuanus. Gallus gallus, Myotis lucifugus, Malaclemys terrapin pileata. Bufo bufo, Zonotrichia leucophrys gambelit. Sorexfumeus, Anolis sagrei. Leopardus geoffroyi, Manacus candei, or Sapajus apella.

[0116] In some embodiments, the tetraspanin poly peptide is derived from human CD81 or CD81 am Xiphophorus couchianus, Lonchura striata, Meriones unguiculatus, Vicugna pacos, Hypanus sabinus, Podarcis muralis, Sinocyclocheilus anshuiensis, Sorex fumeus, Ursus arctos, Myotis davidii, Patagioenas fasciata, Bos taurus, Gopherus flavomarginatus , Orycteropus afer afer, Phasianus colchicus, Atlantic canary7, Florida worm lizard, Adelie penguin, Common toad, or Orbiculate cardinalfish.

[0117] Without being held to theory or mechanism, a tetraspanin polypeptide may comprise an endosome-targeting signal which directs the tetraspanin to the endosome, reducing eVLP formation and eVLP budding. For example, the amino acid sequence of human CD63 has an endosome-targeting signal: YEVM (SEQ ID NO: 208). This YEVM signal directs CD63 to the endosome and cargos in CD63-driven intracellular vesicles usually end up in the endosome. To increase trafficking to the plasma membrane for improved eVLP formation and budding, a modification can be introduced into the endosome-targeting signal. In some embodiments, an EIEIDo described herein comprises a modification in an endosome-targeting signal. In certain embodiments, the endosome-targeting signal that is modified comprises the sequence YEVM.

[0118] In some embodiments, an EIEIDo described herein comprises CD63, or a fragment thereof, comprising a modification in an endosome-targeting signal. In some embodiments, the modification is an amino acid substitution. In certain embodiments, the YEVM sequence in the endosome-targeting signal is modified. In some embodiments, tyrosine (Y) in the YEVM sequence is modified. In certain embodiments, the modification is an amino acid substitution of ty rosine (Y) to alanine (A). In some embodiments, glutamate (E) in the YEVM sequence ismodified. In some embodiments, valine (V) in the YEVM sequence is modified. In some embodiments, methionine (M) in the YEVM sequence is modified.

[0119] In some embodiments, an EIEIDo described herein comprises a CD63 sequence comprising an amino acid substitution at a position corresponding to position 235, 236, 237, and / or 238 relative to SEQ ID NO: 11. In some embodiments, the amino acid substitution is at a position corresponding to position 235 relative to SEQ ID NO: 11. In some embodiments, the amino acid substitution is tyrosine (Y) to alanine (A) at a position corresponding to position 235 relative to SEQ ID NO: 11. Table 2A below provides exemplary amino acid sequences for human CD63 and CD63 from different species with an amino acid substitution in the YEVM endosome-targeting signal sequence, with descriptions providing the position where the amino acid substitution corresponding to position 235 relative to SEQ ID NO: 11 is located in each sequence.

[0120] In some embodiments, an EIEIDo described herein comprises a CD63 sequence comprising the amino acid sequence of any one of SEQ ID NOs: 11 and 168-183, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%. at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%. at least about 85%. at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%. at least about 98%, or at least about 99% sequence identity thereto.

[0121] In some embodiments, the EIEIDos of the disclosure comprise a binding partner of a tetraspanin protein. In some embodiments, without being held to theory or mechanism, the binding partner further aids the tetraspanin proteins (either directly or indirectly) in directing an antigenic polypeptide to an eVLP.

[0122] In some embodiments, the binding partner comprises a human cytomegalovirus (HCMV) protein. In some embodiments, the binding partner comprises the cytoplasmic tail of a HCMV protein. In some embodiments, the HCMV protein is glycoprotein M (gM). In some embodiments, the cytoplasmic tail of a HCMV protein (e.g., gM) co-localize with CD63 (a tetraspanin protein) and direct proteins to eVLPs.

[0123] In some embodiments, an EIEIDo of the disclosure comprises the cytoplasmic tail of HCMV gM. In certain embodiments, the EIEIDos of the disclosure comprise the amino acidsequence of SEQ ID NO: 13 or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%. at least about 84%, at least about 85%, at least about 86%. at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity' thereto.

[0124] Table 2A provides exemplary sequences for tetraspanin polypeptides which may be used in an EIEIDo described herein.Table 2A: Exemplary Tetraspanin Polypeptide or Tetraspanin Binding Partner Amino AcidSequences

[0125] In some embodiments, an EIEIDo described herein comprises at least two tetraspanin polypeptides, or domains or fragments thereof. In some embodiments, the at least two tetraspanin polypeptides are linked together, optionally by a linker, and / or expressed in tandem. In certain embodiments, the at least two tetraspanin polypeptides are linked by a linker. In some embodiments, wherein an EIEIDo described herein comprises at least three tetraspanin polypeptides, the polypeptides are linked by the same linker. In some embodiments, the polypeptides are linked by different linkers. In some embodiments, the linker is a glycine serine linker. In some embodiments, the linker comprises an amino acid sequence selected from Table 8C or Table 8D

[0126] In some embodiments, wherein an EIEIDo described herein comprises at least two tetraspanin polypeptides, the at least two tetraspanin polypeptides comprise the same sequence (e.g., the EIEIDo comprises multiple copies of the same tetraspanin polypeptide), or the at least two tetraspanin polypeptides comprise different sequences (e.g., the EIEIDo comprises a copy of at least two different tetraspanin polypeptides).

[0127] In some embodiments, an EIEIDo described herein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 tetraspanin polypeptides. In certain embodiments, the at least 2, at least 3. at least 4, at least 5, at least 6, at least 7. at least 8, at least 9, or at least 10 tetraspanin polypeptides are the same. In certain embodiments, the at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 tetraspanin polypeptides are different.

[0128] In some embodiments, the at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 tetraspanin polypeptides each comprises an amino acid sequence of Table 2A, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%. at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%. at least about 87%. at least about 88%. at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0129] In some embodiments, the at least 2, at least 3. at least 4, at least 5, at least 6, at least 7. at least 8, at least 9, or at least 10 polypeptides each comprises the amino acid sequence of anyone of SEQ ID NOS: 10-14 and 138-183, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%. at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%. at least about 86%. at least about 87%. at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0130] Also provided herein are engineered variants of tetraspanin polypeptides, also referred to as chimeric tetraspanin polypeptides herein. Chimeric tetraspanin polypeptides were designed by substituting a domain in a first tetraspanin polypeptide with that from a second tetraspanin polypeptide. In some embodiments, an EIEIDo described herein comprises a chimeric tetraspanin polypeptide, wherein the chimeric tetraspanin polypeptide comprises a first tetraspanin polypeptide, wherein a domain in the first tetraspanin polypeptide is substituted with a domain from a second tetraspanin polypeptide. In some embodiments, the domain in the first tetraspanin polypeptide that is substituted is an extracellular domain. In some embodiments, the domain in the second tetraspanin polypeptide used for substitution into the first tetraspanin polypeptide is an extracellular domain. In some embodiments, the first and second tetraspanin polypeptides are independently selected from the group consisting of: CD9, CD63, CD81 , CD82, CD37, CD53, CD151, CD231, tetraspanin (TSP)-l, TSP-2, TSP-3, TSP-4, TSP-5, TSP-6, TSP-9, TSP-11, proteins encoded by genes TSPAN1-TSPAN33, and domains or fragments thereof.

[0131] In some embodiments, the first and second tetraspanin polypeptides are different. In some embodiments, the first and second tetraspanin polypeptides are homologs. In some embodiments, the first and second tetraspanin polypeptides are human tetraspanin polypeptides. In some embodiments, the first and second tetraspanin polypeptides are non-human tetraspanin polypeptides. In some embodiments, the first tetraspanin polypeptide is a human tetraspanin polypeptide and the second tetraspanin polypeptide is anon-human tetraspanin polypeptide. In some embodiments, the first tetraspanin polypeptide is a non-human tetraspanin polypeptide and the second tetraspanin polypeptide is a human tetraspanin polypeptide.

[0132] Table 2B provides exemplary chimeric (Chim) tetraspanin polypeptide sequences. The description of each sequence refers to the name of a first and a second tetraspanin polypeptide.wherein a domain in the first tetraspanin polypeptide is substituted with that of the second tetraspanin polypeptide. For example, CD81-9Chim refers to a chimeric tetraspanin polypeptide comprising a CD81 polypeptide, wherein a domain in CD81 has been substituted with a domain of a CD9 polypeptide. The sequences of Table 2B may be used in an EIEIDo described herein. Although the sequences of Table 2B may be shown to include linkers, these linkers are optional in nature and may be omitted or replaced by other suitable linkers. While * denotes a stop codon in the sequences of Table 2B, such a stop codon is optional in nature and may be omitted.Table 2B: Exemplary Chimeric Tetraspanin Polypeptide Amino Acid Sequences

[0133] In some embodiments, an EIEIDo described herein comprises a tetraspanin polypeptides comprising an amino acid sequence selected from Table 2A, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%. at least about 75%, at least about 76%, at least about 77%, at least about 78%. at least about 79%. at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%. at least about 98%. or at least about 99% sequence identity thereto. In some embodiments, the tetraspanin polypeptide comprise the amino acid sequence of any one of SEQ ID NOS: 10- 13, 138-183, and 218-227, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%. at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%. at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0134] In some embodiments, an EIEIDo described herein comprising at least two tetraspanin polypeptides comprises an amino acid sequence selected from Table 2A or Table 2B, or an amino acid sequence having at least about 70%, at least about 71%. at least about 72%, at least about 73%. at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%. at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%. at least about 98%. or at least about 99% sequence identity thereto. In some embodiments, an EIEIDo described herein comprising at least two tetraspanin polypeptides comprises the amino acid sequence of any one of SEQ ID NOS: 10-14, 138-207, and 218-227, or an amino acid sequence having at least about 70%, at least about 71%. at least about 72%, at least about 73%, at least about 74%. at least about 75%. at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%. at least about 98%, or at least about 99% sequence identity thereto.

[0135] In some embodiments, an ElElDo described herein comprising a chimeric tetraspanin polypeptide comprises an amino acid sequence selected from Table 2B, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%. at least about 75%, at least about 76%, at least about 77%, at least about 78%. at least about 79%. at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99% sequence identity thereto. In some embodiments, an EIEIDo described herein comprising a chimeric tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 184-207, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%. at least about 78%, at least about 79%, at least about 80%. at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0136] Also disclosed herein are polypeptides comprising an EIEIDo described herein comprising a tetraspanin polypeptide (including naturally occurring tetraspanin polypeptides or variants thereof), or a domain or fragment thereof, or a binding partner thereof. It can be appreciated that any EIEIDo described herein comprising a tetraspanin polypeptide or a chimeric tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof, may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, which are further discussed herein in detail. Provided herein are polypeptides comprising a sequence in Table 2A or Table 2B.

[0137] Additionally, disclosed herein are polynucleotides encoding the polypeptides comprising an EIEIDo described herein comprising a tetraspanin polypeptide, or a domain or fragmentthereof, or a binding partner thereof and polynucleotides encoding fusion proteins comprising an EIEIDo described herein comprising a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof. In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprises an antigenic polypeptide described herein and an EIEIDo described herein comprising a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof. Provided herein are polynucleotides that encode any one or more of the sequences of Table 2A and Table 2B, or a sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%. at least about 74%, at least about 75%, at least about 76%. at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%. at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0138] Polynucleotides can be DNA or RNA (e.g., mRNA), as described in detail herein.LAMP2 and ExoSignal polypeptides

[0139] In some embodiments, the ESCRT-independent enveloped virus-like particle (eVLP) inducing domain (EIEIDo) comprises a lysosome-associated membrane protein 2 (LAMP2) polypeptide, or a domain or fragment thereof, or a binding partner thereof. As used herein, the term “LAMP 2 polypeptide’' is all encompassing, and inclusive of naturally occurring LAMP2 polypeptides and engineered variants thereof. LAMP2 is an exosomal protein that comprises three variants: Lamp2A, Lamp2B, and Lamp2C. Without being held to theory or mechanism, LAMP2 may directly bind to proteins that are sorted into an eVLP or may redirect proteins into an eVLP. In some embodiments, when expressed with an antigenic polypeptide, the LAMP2 polypeptide, or a domain or fragment thereof, or a binding partner thereof, facilitates generation of an eVLP with the antigenic polypeptide extended therefrom. In some embodiments, the binding partner of the LAMP2 polypeptide comprises an ExoSignal. In certain embodiments, the ExoSignal associates with a LAMP2 polypeptide and / or facilitates generation of eVLP.

[0140] FIGS. 3A-3B and FIG. 4 provide cartoon schematics of fusion proteins comprising a LAMP2 polypeptide or an ExoSignal coupled to antigenic polypeptides in an eVLP, which are further exemplified in Example 1.

[0141] In some embodiments, an EIEIDo described herein comprises a Lamp2A polypeptide or a Lamp2B polypeptide. In some embodiments, an EIEIDo described herein comprises a LAMP2 polypeptide, or fragments thereof, comprising an amino acid sequence selected from Table 3. In some embodiments, an EIEIDo described herein comprises a sequence derived from human Lamp2A. Lamp2B. Lamp2C, or a fragment thereof. Alternatively, homologous Lamp2A, Lamp2B, Lamp2C polypeptides from other species may be utilized. In some embodiments, homologous Lamp2A, Lamp2B, or Lamp2C polypeptides from vertebrates or invertebrates are utilized. In some embodiments, an EIEIDo described herein comprises a sequence derived from a Lamp2A. Lamp2B, or Lamp2C polypeptide from a non-human vertebrate or an invertebrate.

[0142] In some embodiments, the LAMP2 polypeptide comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%. at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%. at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0143] In some embodiments, an EIEIDo described herein comprises a binding partner of LAMP2. In some embodiments, without being held to theory' or mechanism, the binding partner associates with a LAMP2 polypeptide. In some embodiments, the binding partner comprises a sequence (e.g., an ExoSignal) that associates with a LAMP2 protein. In some embodiments, the binding partner comprises an ExoSignal. In some embodiments, the binding partner of LAMP2 (e.g., an ExoSignal) is fused to the cytoplasmic end of an antigenic polypeptide. In some embodiments, an EIEIDo described herein comprises at least 2, at least 3, at least 4, or at least 5 copies of a sequence (e.g., an ExoSignal) that associates with a LAMP2 protein. In some embodiments, an EIEIDo described herein comprises 1. 2, 3, 4. or 5 copies of a sequence (e.g., an ExoSignal) that associates with a LAMP2 protein. In some embodiments, the sequence (e.g., an ExoSignal) that associates with a LAMP2 protein comprises the amino acid sequence of any one of SEQ ID NOS: 18-20, or a fragment thereof. In some embodiments, the sequence (e.g.. an ExoSignal) that associates with a LAMP2 protein comprises an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%,at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%. at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 18-20.

[0144] In some embodiments, a binding partner of a LAMP2 polypeptide comprises a motif. Without being held to theory or mechanism, the binding partner may comprise a motif configured to help LAMP2 polypeptides redirect proteins to eVLPs. In some embodiments, the motif comprises the amino acid sequence KFERQ (SEQ ID NO: 17). In some embodiments, an EIEIDo described herein comprises at least 2, at least 3, at least 4, or at least 5 copies of a motif configured to help LAMP2 polypeptides redirect proteins to eVLPs. In some embodiments, an EIEIDo described herein comprises 1, 2, 3, 4, or 5 copies of a motif configured to help LAMP2 polypeptides redirect proteins to eVLPs. In some embodiments, an EIEIDo described herein comprises at least 2, at least 3, at least 4, or at least 5 motifs configured to help LAMP2 polypeptides redirect proteins to eVLPs. In some embodiments, an EIEIDo described herein comprises 1, 2. 3, 4, or 5 motifs configured to help LAMP2 polypeptides redirect proteins to eVLPs. In some embodiments, an EIEIDo described herein comprises at least 2, at least 3. at least 4, or at least 5 copies of 1 , 2, 3, 4, or 5 motifs configured to help LAMP2 polypeptides redirect proteins to eVLPs.

[0145] Table 3 provides exemplary sequences for LAMP2 polypeptides, or a domain or fragment thereof, or a binding partner thereof, which may be used in an EIEIDo described herein.Table 3: Exemplary LAMP2 Amino Acid Sequences

[0146] In some embodiments, an EIEIDo described herein comprises a lysosome-associated membrane protein 2 (LAMP2) polypeptide, or a domain or fragment thereof, or a binding partner thereof comprising an amino acid sequence selected from Table 3, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%. at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%. at least about 98%. or at least about 99% sequence identity thereto. In some embodiments, the LAMP2 polypeptide, or a domain or fragment thereof, or a binding partner thereof comprises the amino acid sequence of any one of SEQ ID NOS: 15-20, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%. at least about 75%. at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0147] Also disclosed herein are polypeptides comprising an EIEIDo described herein comprising a LAMP2 polypeptide, or a domain or fragment thereof, or a binding partner thereof. It can be appreciated that any EIEIDo described herein comprising a LAMP2 polypeptide, or a domain or fragment thereof, or a binding partner thereof, may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, which are further discussed herein in detail. Provided herein are polypeptides comprising a sequence in Table 3.

[0148] Additionally, disclosed herein are polynucleotides encoding the polypeptides comprising an EIEIDo described herein comprising a LAMP2 polypeptide, or a domain or fragment thereof, or a binding partner thereof, and polynucleotides encoding fusion proteins comprising an EIEIDo described herein comprising a LAMP2 polypeptide, or a domain or fragment thereof, or a binding partner thereof. In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprises an antigenic polypeptide described herein and an EIEIDo described herein comprising a LAMP2 polypeptide, or a domain or fragment thereof, or a binding partner thereof. Provided herein are polynucleotides that encode any one or more of the sequences of Table 3, or a sequence having at least about 70%, at least about 71%, at least about 72%. at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%. at least about 89%, at least about 90%, at least about 91%. at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0149] Polynucleotides can be DNA or RNA (e g., mRNA), as described in detail herein.Phosphatidylserine-binding polypeptides

[0150] In some embodiments, an EIEIDo described herein comprises a polypeptide that binds to phosphatidylserine (a phosphatidylserine-binding polypeptide). As used herein, the term “polypeptide that binds to phosphatidylserine'’ is all encompassing, and inclusive of naturally occurring phosphatidylserine-binding polypeptides and engineered variants thereof. In some embodiments, the polypeptide that binds to phosphatidylserine comprises an amino acid sequence selected from Table 4 below. In some embodiments, the polypeptide that binds tophosphatidylserine comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%. at least about 80%, at least about 81%, at least about 82%, at least about 83%. at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99% sequence identity thereto.

[0151] In some embodiments, the polypeptide that binds to phosphatidylserine comprises an antibody or antibody fragment thereof with specificity for phosphatidylserine.

[0152] In some embodiments, the polypeptide that binds to phosphatidylserine comprises a lactadherin polypeptide. As used herein, the term “lactadherin polypeptide'’ is all encompassing, and inclusive of naturally occurring lactadherin polypeptides and engineered variants thereof. In some embodiments, an EIEIDo described herein comprises a lactadherin polypeptide. In some embodiments, the lactadherin polypeptide comprises a full length lactadherin polypeptide, one or more domains of a lactadherin polypeptide, or one or more fragments of a lactadherin polypeptide. In some embodiments, when expressed with an antigenic polypeptide, the phosphatidyl serine-binding polypeptide (e.g., lactadherin polypeptide) facilitates generation of an eVLP with the antigenic polypeptide extended therefrom. FIG. 5 provides a cartoon schematic of phosphatidylserines associating with fusion proteins comprising a lactadherin polypeptide, or domain or fragment thereof, coupled to antigenic polypeptides in an eVLP.

[0153] In some embodiments, the lactadherin polypeptide comprises a C1C2 domain of lactadherin. In certain embodiments, the C1C2 domain comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having about 70% sequence identity thereto. For example, in some embodiments, the lactadherin polypeptide comprises an amino acid sequence having at least about 70%, at least about 71%. at least about 72%, at least about 73%, at least about 74%. at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%. at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0154] In some embodiments, an EIEIDo described herein comprises a sequence derived from a human lactadherin polypeptide, or a domain or fragment thereof. Alternatively, homologous lactadherin polypeptides from other species may be utilized. In some embodiments, homologous lactadherin polypeptides from vertebrates or invertebrates are utilized. In some embodiments, an EIEIDo described herein comprises a sequence derived from a lactadherin polypeptide from a non-human vertebrate or an invertebrate.

[0155] In some embodiments, the lactadherin polypeptide comprises an amino acid sequence selected from Table 4 below. In some embodiments, the lactadherin polypeptide comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having about 70% sequence identity thereto. For example, in some embodiments, the lactadherin polypeptide comprises an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%. at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%. at least about 80%. at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0156] Table 4 provides an exemplary sequence for a lactadherin polypeptide which may be used in an EIEIDo described herein.Table 4: Exemplary Lactadherin Amino Acid Sequence

[0157] In some embodiments, an EIEIDo described herein comprises a polypeptide that binds to phosphatidylserine (e.g., a lactadherin polypeptide), or a domain or fragment thereof, comprising an amino acid sequence selected from Table 4, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%,at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%. at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the polypeptide that binds to phosphatidylserine (e.g., a lactadherin polypeptide), or a domain or fragment thereof, comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least about 70%, at least about 71%. at least about 72%. at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%. at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%. at least about 91%. at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0158] Also disclosed herein are polypeptides comprising an EIEIDo described herein comprising a phosphatidylserine-binding polypeptide (e.g., a lactadherin polypeptide). It can be appreciated that any EIEIDo described herein comprising a phosphatidylserine-binding polypeptide (e.g., a lactadherin polypeptide) may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, which are further discussed herein in detail. Provided herein are polypeptides comprising a sequence in Table 4.

[0159] Additionally, disclosed herein are polynucleotides encoding the polypeptides comprising an EIEIDo described herein comprising a phosphatidylserine-binding polypeptide (e.g., a lactadherin polypeptide) and polynucleotides encoding fusion proteins comprising an EIEIDo described herein comprising a phosphatidylserine-binding polypeptide (e.g., a lactadherin polypeptide). In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprises an antigenic polypeptide described herein and an EIEIDo described herein comprising a phosphatidylserine-binding polypeptide (e.g., a lactadherin polypeptide). Provided herein are polynucleotides that encode any one or more of the sequences of Table 4, or a sequence having at least about 70%, at least about 71%, at least about 72%. at least about 73%. at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, atleast about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0160] Polynucleotides can be DNA or RNA (e g., mRNA), as described in detail herein.PDGFR polypeptide

[0161] In some embodiments, an EIEIDo described herein comprises a Platelet-Derived Grow th Factor Receptor (PDGFR) polypeptide, or a domain or fragment thereof. As used herein, the term “PDGFR polypeptide” is all encompassing, and inclusive of naturally occurring PDGFR polypeptides and engineered variants thereof. Without being held to theory or mechanism, the PDGFR polypeptide, or a domain or fragment thereof, may direct proteins into eVLPs. In some embodiments, when expressed with an antigenic polypeptide, the PDGFR polypeptide facilitates the generation of an eVLP with the antigenic polypeptide extended therefrom. FIG. 6 depicts a cartoon schematic of fusion proteins comprising a PDGFR polypeptide coupled to antigenic polypeptides in an eVLP.

[0162] In some embodiments, the PDGFR polypeptide comprises a full length PDGFR polypeptide. In some embodiments, the PDGFR polypeptide comprises a domain or fragment of a full length PDGFR polypeptide. In some embodiments, an EIEIDo described herein comprises a sequence derived from a human PDGFR polypeptide, or a domain or fragment thereof. Alternatively, homologous PDGFR polypeptides from other species may be utilized. In some embodiments, homologous PDGFR polypeptides from vertebrates or invertebrates are utilized. In some embodiments, an EIEIDo described herein comprises a sequence derived from a PDGFR polypeptide from a non-human vertebrate or an invertebrate. Example of non-human vertebrates or invertebrates from which a PDGFR polypeptide can be derived include, but are not limited to, Spanish mole, brushtail possum, western clawed frog, Chinese alligator, and common sole fish. In certain embodiments, an EIEIDo described herein comprises a sequence derived from a PDGFR poly peptide of a Spanish mole. In certain embodiments, an EIEIDo described herein comprises a sequence derived from a PDGFR polypeptide of a brushtail possum. In certain embodiments, an EIEIDo described herein comprises a sequence derived from a PDGFR polypeptide of a western clawed frog. In certain embodiments, an EIEIDo described herein comprises a sequence derived from a PDGFR polypeptide of a Chinesealligator. In certain embodiments, an EIEIDo described herein comprises a sequence derived from a PDGFR polypeptide of a common sole fish.

[0163] In some embodiments, the PDGFR polypeptide comprises the transmembrane domain of a PDGFR polypeptide. In some embodiments, the PDGFR polypeptide comprises an amino acid sequence selected from Table 5. In some embodiments, the PDGFR polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 22 and 228-232, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%. at least about 75%, at least about 76%, at least about 77%, at least about 78%. at least about 79%. at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99% sequence identity thereto.

[0164] Table 5 provides an exemplary sequence for a PDGFR polypeptide which may be used in an EIEIDo described herein.Table 5: Exemplary PDGFR transmembrane domain Amino Acid Sequence

[0165] In some embodiments, an EIEIDo described herein comprises a PDGFR polypeptide, or a domain or fragment thereof, comprising an amino acid sequence selected from Table 5, or an amino acid sequence having at least about 70%, at least about 71%. at least about 72%, at least about 73%. at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the PDGFR polypeptide, or a domain or fragment thereof, comprises the amino acid sequence of any one of SEQ ID NOS: 22 and 228-232. or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%. at least about 81%, at least about 82%, at least about 83%, at least about 84%. at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0166] Also disclosed herein are polypeptides comprising an EIEIDo described herein comprising a PDGFR polypeptide. It can be appreciated that any EIEIDo described herein comprising a PDGFR polypeptide may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, which are further discussed herein in detail. Provided herein are polypeptides comprising a sequence in Table 5.

[0167] Additionally, disclosed herein are polynucleotides encoding the polypeptides comprising an EIEIDo described herein comprising a PDGFR polypeptide and polynucleotides encoding fusion proteins comprising an EIEIDo described herein comprising a PDGFR polypeptide. In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprises an antigenic polypeptide described herein and an EIEIDo described herein comprising a PDGFR polypeptide. Provided herein are polynucleotides that encode any one or more of the sequences of Table 5, or a sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%. at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%. at least about 82%. at least about 83%. at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%. at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0168] Polynucleotides can be DNA or RNA (e.g., mRNA), as described in detail herein.Polypeptide capable of forming an amphipathic helix

[0169] In some embodiments, an EIEIDo described herein comprises a polypeptide capable of forming an amphipathic helix, or a domain or fragment thereof. As used herein, the term “polypeptide capable of forming an amphipathic helix” is all encompassing, and inclusive of naturally occurring polypeptides capable of forming an amphipathic helix and engineered variants thereof, some embodiments, the amphipathic helix comprises an alpha helix with hydrophobic and hydrophilic amino acid residues arranged to create two faces on opposite sides of the helix, wherein one face is hydrophobic, and one face is hydrophilic. In some embodiments, an EIEIDo described herein comprising a polypeptide capable of forming an amphipathic helix comprises hydrophobic and hydrophilic amino acid residues arranged to create two faces on opposite sides of the amphipathic helix, wherein one face is hydrophobic, and one face is hydrophilic.

[0170] Without being held to theory or mechanism, polypeptides capable of forming an amphipathic helix may be configured to induce membrane fission and / or increase membrane curvature. In some embodiments, an EIEIDo described herein comprising a polypeptide capable of forming an amphipathic helix induces membrane fission and / or increases membrane curvature when expressed in a cell.

[0171] In some embodiments, when expressed with an antigenic polypeptide, the polypeptide capable of forming an amphipathic helix facilitates generation of an eVLP with a distinct curvature, with the antigenic polypeptide extended therefrom. In some embodiments, the antigenic polypeptide and the polypeptide capable of forming an amphipathic helix are sourced from unique polypeptides, wherein the antigenic polypeptide comprises a different polypeptide capable of forming an amphipathic helix.

[0172] In some embodiments, the polypeptide capable of forming an amphipathic helix, when expressed in a cell, promotes membrane curvature and therefore budding of eVLPs from the surface of the cell. Without being held to theory or mechanism, this membrane curvature may be due to “warping” of the hydrophobic membrane, promoted by the hydrophobic membrane's interactions with the hydrophobic residues of the polypeptide capable of forming an amphipathic helix. FIGS. 7A-7C depict cartoon schematics of eVLPs with antigenic polypeptides coupled to a polypeptide capable of forming an amphipathic helix.

[0173] In some embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from the M2 protein of influenza A, monoglucosyldiacylglycerol synthase(MGS) from Acholeplasma laidlawii, septum site-determining protein MinD from a bacterium (e.g., E. coli). peroxisomal membrane protein Peroxin 11 (Pexl Ip), phospholipid N- methyltransferase PmtA from Agrobacterium tumefaciens (AtPmtA), N-acetylmuramyl- (pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (MurG) from A. coli (EcMurG), EH domain-binding mitotic phosphoprotein (Epsin 1), amphiphysin from Drosophila, protein interacting with C kinase 1 (PICK1), ADP-ribosylation factor 1 (Arfl), diglucosyldiacylglycerol synthase (DGS) from Acholeplasma laidlawii, Endophilin Al, mammalian amphiphysin 2, Clathrin Assembly Lymphoid-Myeloid leukemia protein (CALM), or alpha-synuclein (a-synuclein).

[0174] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from M2 protein of influenza A.

[0175] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from MinD protein from a bacterium (e.g., E. coli). In specific embodiments, the bacterium is E. coli.

[0176] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from Pexl Ip.

[0177] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from AtPmtA.

[0178] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from EcMurG.

[0179] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from Epsin 1.

[0180] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from amphiphysin from Drosophila.

[0181] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from PICK1.

[0182] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from Arfl .

[0183] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from DGS from Acholeplasma laidlawii.

[0184] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from Endophilin AL

[0185] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from mammalian amphiphysin 2.

[0186] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from CALM.

[0187] In certain embodiments, the polypeptide capable of forming an amphipathic helix is isolated or derived from a-synuclein.

[0188] In some embodiments, the polypeptide capable of forming an amphipathic helix comprises an amino acid sequence selected from Table 6. In some embodiments, the polypeptide capable of forming an amphipathic helix comprises the amino acid sequence of any one of SEQ ID NOS: 23-39, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%. at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%. at least about 83%. at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0189] Table 6 provides exemplary sequences for polypeptides capable of forming an amphipathic helix which may be used in an EIEIDo described herein. MGS denotes monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii, AtPmtA denotes PmtA from Agrobacterium tumefaciens. MinD denotes septum site-determining protein from A. coli. Pexl Ip denotes peroxisomal membrane protein Peroxin 11. EcMurG denotes MurG from E. coli. PICK1 denotes protein interacting with C kinase 1. Arfl denotes ADP-ribosylation factor 1. DGS denotes diglucosyldiacylglycerol synthase. CALM denotes Clathrin Assembly Lymphoid- Myeloid leukemia protein. M denotes M2 protein of influenza A. TM denotes transmembrane domain, and CT denotes cytoplasmic tail.Table 6: Exemplary Amphipathic Helix Amino Acid Sequences

[0190] In some embodiments, an ElElDo described herein comprising a polypeptide capable of forming an amphipathic helix comprises an amino acid sequence of Table 6, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%. at least about 75%, at least about 76%, at least about 77%, at least about 78%. at least about 79%. at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99% sequence identity thereto.

[0191] In some embodiments, an EIEIDo described herein comprises at least two polypeptides, wherein each polypeptide is capable of forming an amphipathic helix. In some embodiments, the at least two polypeptides are linked together, optionally by a linker, and / or expressed in tandem. FIGS. 15A-15C depict schematics of amphipathic helices coupled together by a linker, and FIGS. 15D-15F depict schematics of amphipathic helices expressed in tandem (‘"in frame’7). In some embodiments, wherein an EIEIDo described herein comprises at least two polypeptides capable of forming an amphipathic helix, the at least two polypeptides comprise the same sequence (e.g., the EIEIDo comprises multiple copies of the same polypeptide capable of forming an amphipathic helix), or the at least two polypeptides comprise different sequence(e.g., the EIEIDo comprises a copy of at least two different polypeptide, each capable of forming an amphipathic helix).

[0192] In some embodiments, the at least two polypeptides capable of forming an amphipathic helix are linked by a linker. In some embodiments, the linker links the C-terminus of one polypeptide to the N-terminus of another polypeptide. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids in length. In certain embodiments, the linker is 8 amino acids in length. In some embodiments, the linker is a glycine serine linker. In some embodiments, the length of the linker is about 1 / 5 to 1 / 3 of the number of amino acids of one of the at least two polypeptides capable of forming an amphipathic helix. In certain embodiments, the length of the linker is about 1 / 5 of the number of amino acids of one of the at least two polypeptides capable of forming an amphipathic helix. In certain embodiments, the length of the linker is about 1 / 4 of the number of amino acids of one of the at least two polypeptides capable of forming an amphipathic helix. In certain embodiments, the length of the linker is about 1 / 3 of the number of amino acids of one of the at least two polypeptides capable of forming an amphipathic 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 to the N-terminus of the other alpha helix while allowing the formation of a 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 at least two polypeptides capable of forming an amphipathic 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 at least two polypeptides capable of forming an amphipathic helix.

[0193] The at least two polypeptides capable of forming an amphipathic helix may be truncated, thereby allowing for the use of a shorter linker to connect the sequences while allowing for the formation of two alpha helices and a coiled-coil structure. In some embodiments, the linker is a glycine serine linker. In some embodiments, the linker comprises an amino acid sequence selected from Table 8C. In some embodiments, wherein an EIEIDo described herein comprises at least three polypeptides capable of forming an amphipathic helix, the polypeptides are linked by the same linker. In some embodiments, the polypeptides are linked by different linkers.

[0194] As examples, the design of polypeptides capable of forming an amphipathic helix is depicted in FIGS. 17A-17C. As illustrated in the schematic of FIG. 17A, an alpha helix formswith repeat units of 7 residues, such that position 1 and position 8 are aligned, correlating to a traditional helical wheel representation shown below the helix representation. FIGS. 17B-17C are schematics depicting traditional helical wheel representations of 2 exemplary polypeptides capable of forming amphipathic helices, w hich are described in further detail in this disclosure and were tested experimentally. Hydrophobic regions are highlighted as dashed lines and the sequence and corresponding residue numbers of the 7 positions in the helical w heel are shown below the wheel representations. Tandem sequences for two exemplary' polypeptides capable of forming amphipathic helices, EcMurG (FIG. 17B) and CALM (FIG. 17C) are shown, with extensions of positions 5, 6, and 7 in the helical wheel shown to maintain the "frame" of the helix for subsequent extensions. The proline residue in position 1 in the EcMurG sequence may be replaced by an alanine to remove the “helix breaker” residue.

[0195] In some embodiments, an EIEIDo described herein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9. or at least 10 polypeptides capable of forming an amphipathic helix. In certain embodiments, the at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 polypeptides are the same. In certain embodiments, the at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 polypeptides are different.

[0196] In some embodiments, at least 2. at least 3. at least 4, at least 5, at least 6, at least 7. at least 8, at least 9, or at least 10 polypeptides each comprises the amino acid sequence of any one of SEQ ID NOS: 23-39, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%. at least about 73%, at least about 74%, at least about 75%, at least about 76%. at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%. at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity' thereto. In some embodiments, the at least 2, at least 3. at least 4. at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 polypeptides each comprises the amino acid sequence of any one of SEQ ID NOS: 41-120, or an amino acid sequence having at least about 70%, at least about 71%. at least about 72%, at least about 73%, at least about 74%, at least about 75%. at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%,at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0197] Table 7 provides exemplary sequences for at least two polypeptides linked together, wherein each polypeptide is capable of forming an amphipathic helix. The source of the polypeptides linked together are identified in the construct description and the sequences of Table 7 may be used in an EIEIDo described herein. Although the sequences of Table 7 may be shown to include linkers, these linkers are optional in nature and may be omitted or replaced by other suitable linkers. In the descriptions of Table 7, unless otherwise indicated, MGS denotes monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii, AtPmtA denotes PmtA from Agrobacterium tumefaciens . MinD denotes septum site-determining protein from A. coli. Pexl Ip denotes peroxisomal membrane protein Peroxin 11. EcMurG denotes MurG from E. coli. PICK1 denotes protein interacting with C kinase 1. Arfl denotes ADP-ribosylation factor 1. DGS denotes diglucosyldiacylglycerol synthase. CALM denotes Clathrin Assembly Lymphoid- Myeloid leukemia protein. M2 denotes M2 protein of influenza A. TM denotes transmembrane domain, and CT denotes cytoplasmic tail.; “x3” denotes 3 copies of the same polypeptide linked together; and “x5” denotes 5 copies of the same polypeptide linked together; “cont7’ denotes multiple copies of a given amphipathic helix linked in frame without a linker.

[0198] In some embodiments, an EIEIDo described herein comprising a polypeptide capable of forming an amphipathic helix comprises an amino acid sequence of Table 7, or an amino acid sequence having at least about 70%. at least about 71%. at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%. at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, an EIEIDo described herein comprising at least two polypeptides, wherein each of the at least two polypeptides is capable of forming an amphipathic helix, comprises the amino acid sequence of any one of SEQ ID NOS: 41-120, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at leastabout 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%. at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.Table 7: Exemplary Amino Acid Sequences of Two or more Amphipathic Helicesforming an amphipathic helix comprises an amino acid sequence selected from Table 6 or Table 7, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%. at least about 74%, at least about 75%, at least about 76%, at least about77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%. at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the polypeptide capable of forming an amphipathic helix comprises the amino acid sequence of any one of SEQ ID NOS: 23-120, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%. at least about 73%, at least about 74%, at least about 75%. at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at leastabout 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0200] In some embodiments, an ElElDo described herein comprising at least two polypeptides, wherein each of the at least two polypeptides is capable of forming an amphipathic helix, comprises an amino acid sequence selected from Table 6 or Table 7, or an amino acid sequence having at least about 70%, at least about 71%. at least about 72%, at least about 73%, at least about 74%. at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, an EIEIDo described herein comprising at least two polypeptides, wherein each of the at least two polypeptides is capable of forming an amphipathic helix, comprises the amino acid sequence of any one of SEQ ID NOS: 23-120, or an amino acid sequence having at least about 70%. at least about 71%. at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%. at least about 90%. at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0201] Also disclosed herein are polypeptides comprising an EIEIDo described herein comprising a polypeptide capable of forming an amphipathic helix. It can be appreciated that any EIEIDo described herein comprising a polypeptide capable of forming an amphipathic helix may be included in combination with an antigenic polypeptide to generate the fusion proteins described herein, which are further discussed herein in detail. Provided herein are polypeptides comprising a sequence in Table 6 or Table 7.

[0202] Additionally, disclosed herein are polynucleotides encoding an EIEIDo described herein comprising a polypeptide capable of forming an amphipathic helix and polynucleotides encodingthe fusion proteins comprising an EIEIDo described herein comprising a polypeptide capable of forming an amphipathic helix. In some embodiments, the polynucleotides of the disclosure encode a fusion protein, wherein the fusion protein comprises an antigenic polypeptide described herein and an EIEIDo described herein comprising a polypeptide capable of forming an amphipathic helix. Provided herein are polynucleotides that encode any one or more of the sequences of Table 6 or Table 7, or a sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%. at least about 78%, at least about 79%, at least about 80%, at least about 81%. at least about 82%. at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0203] Polynucleotides can be DNA or RNA (e.g., mRNA), as described in detail herein.PDZ domain

[0204] As used herein, a PDZ domain refers to a common structural domain typically about 80-90 amino acids in length. PDZ domains are common to scaffold and signaling proteins and play an important role in signal transduction complexes. PDZ domains of a protein can facilitate protein-protein interactions by binding target proteins. In some embodiments, an EIEIDo of the disclosure comprises a PDZ domain. Without being held to theory or mechanism, in some embodiments, the inclusion of a PDZ domain facilitates colocalization of a polypeptide which binds to the PDZ domain with antigenic polypeptides which associate with or are directly fused to the EIEIDo. Inclusion of a PDZ domain can therefore facilitate display of the polypeptide which binds to the PDZ domain on the surface of eVLPs whose formation is driven by the EIEIDo. For example, an EIEIDo of the disclosure, when expressed w ith an antigenic polypeptide, facilitates formation of eVLPs which display on their surface the antigenic polypeptide, or a portion thereof. An EIEIDo comprising a PDZ domain, when expressed with an antigenic polypeptide, facilitates formation of eVLPs which display on their surface the antigenic polypeptide, or a portion thereof, as well as polypeptides which bind the PDZ domain. In some embodiments, an EIEIDo of the disclosure comprises a PDZ domain, thereby facilitating formation of eVLPs which display on their surface polypeptides which bind the PDZdomain. In some embodiments, a PDZ domain is fused to the C-terminus of a tetraspanin polypeptide to promote MHC association.

[0205] In some embodiments, an EIEIDo of the disclosure comprises a PDZ1 domain. In some embodiments, a fusion protein of the disclosure comprises a PDZ1 domain. Major Histocompatibility Complex class I (MHC class 1) has a PDZ1 binding domain and therefore binds to polypeptides with a PDZ1 domain through interaction with the PDZ1 domain. Thus, in some embodiments where an EIEIDo of the disclosure comprises a PDZ1 domain, the EIEIDo facilitates formation of eVLPs which display on their surface MHC class I and antigenic polypeptides associated with or fused directly to the EIEIDo. Exemplary EIEIDo sequences comprising a PDZ1 domain are provided in Table 2B as SEQ ID NOS: 206 and 207. In some embodiments, an EIEIDo of the disclosure comprises the PDZ1 domain sequence within the amino acid sequences of SEQ ID NO: 206 or SEQ ID NO: 207. or an amino acid sequence having at least about 70%, at least about 71%. at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%. at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, an EIEIDo of the disclosure comprises a PDZ1 domain comprising the amino acid sequence PLGSMEELTLTILRQTGGLGISIAGGKGSTPYKGDDEGIFISRVSEEGPAARAGVRVGDK LLEVNGVALQGAEHHEAVEALRGAGTAVQMRVWRERETSV (SEQ ID NO: 235), or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%. at least about 84%, at least about 85%, at least about 86%, at least about 87%. at least about 88%. at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.Endocytosis Prevention Motifs

[0206] In some embodiments, an ESCRT-independent eVLP inducing domains (EIEIDo) described herein, a polypeptide described herein, or a fusion protein described herein comprises an endocytosis prevention motif (EPM). In some embodiments, without being held to theory or mechanism, the EPM decreases the rate of endocytosis of polypeptides, thereby providing antigenic polypeptides an increased chance to bud at the cell surface. In some embodiments, the EPM increases eVLP formation when expressed in a cell.

[0207] In some embodiments, the EPM comprises the amino acid sequence of ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY (SEQ ID NO: 237), or an amino acid sequence having at least about 70%, at least about 71%. at least about 72%, at least about 73%. at least about 74%. at least about 75%. at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%. at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.Fusion Proteins

[0208] In an aspect, disclosed herein is a fusion protein comprising an antigenic poly peptide described herein and an ESCRT-independent eVLP inducing domain (EIEIDo) described herein. In some embodiments, a fusion protein of the disclosure comprises an EIEIDo compnsing: (1) an envelope protein of a virus, or a domain or fragment thereof; (2) a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof; (3) a lysosome-associated membrane protein 2 (LAMP2) polypeptide, or a domain or fragment thereof, or a binding partner thereof; (4) a polypeptide that binds to phosphatidylserine, or a domain or fragment thereof; (5) a platelet-derived growth factor receptor (PDGFR) polypeptide, or a domain or fragment thereof; or (6) a polypeptide capable of forming an amphipathic helix. Without being bound to theory or mechanism, it is thought that the EIEIDo of a fusion protein of the disclosure facilitates production of eVLPs in a manner not dependent on ESCRT machinery of a cell.

[0209] In some embodiments, a fusion protein of the disclosure comprises an antigenic polypeptide derived from an antigen in Table 8A below. In some embodiments, a fusion protein of the disclosure comprises an antigenic polypeptide comprising an amino acid sequenceselected from Table 8B below. In some embodiments, the antigenic polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 1-5, or an amino acid sequence having at least 70% sequence identity thereto. In some embodiments, a fusion protein of the disclosure comprises an EIEIDo that facilitates production of eVLPs and comprises the amino acid sequence of any one of SEQ ID NOS: 6-120, or an ammo acid sequence having at least about 70% sequence identity thereto.

[0210] In some embodiments, a fusion protein of the disclosure comprises one or more EIEIDos described herein comprising an amino acid sequence of Table 1, Table 2A, Table 2B, Table 3, Table 4, Table 5, Table 6. or Table 7, 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.

[0211] In some embodiments, a fusion protein of the disclosure comprises one or more EIEIDos described herein comprising an amino acid sequence of SEQ ID NOs: 6-120, 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.

[0212] In some embodiments, a fusion protein of the disclosure comprises a substitution of a transmembrane domain of the antigenic polypeptide to reduce spontaneous release of the fusion protein when expressed in a cell. In certain embodiments, the transmembrane domain of the antigenic polypeptide is substituted for a transmembrane domain from Epstein-Barr Virus (EBV) gp220 protein, Sendai Virus F protein, SARS-CoV-2 E protein, or Human Cytomegalovirus Virus gH protein. In some embodiments, the antigenic polypeptide is NS1 from a flavivirus. In some embodiments, the antigenic polypeptide is WNV NS1.

[0213] In some embodiments, a fusion protein described herein comprises an endocytosis prevention motif (EPM), a signal peptide, and / or a transmembrane domain. In some embodiments, a fusion protein of the disclosure comprises an EPM. In some embodiments, a fusion protein of the disclosure comprises a signal peptide. In some embodiments, a fusionprotein of the disclosure comprises a transmembrane domain. In some embodiments, a fusion protein of the disclosure comprises at least 2, at least 3, at least 4, or at least 5 transmembrane domains. In some embodiments, a fusion protein of the disclosure comprises 1, 2, 3, 4, or 5 transmembrane domains.

[0214] In some embodiments, a fusion protein of the disclosure comprises, from N-terminus to C-terminus, an antigenic polypeptide and an EIEIDo. In some embodiments, a fusion protein of the disclosure comprises, from N-terminus to C-terminus, an antigenic polypeptide, an EIEIDo, and an EPM. In some embodiments, a fusion protein of the disclosure comprises, from N- terminus to C-terminus, an antigenic polypeptide, an EPM, and an EIEIDo.

[0215] In some embodiments, a fusion protein of the disclosure comprises, from N-terminus to C-terminus, an antigenic polypeptide, a transmembrane domain, and an EIEIDo. In some embodiments, a fusion protein of the disclosure comprises, from N-terminus to C-terminus, an antigenic polypeptide, a transmembrane domain, an EIEIDo, and an EPM. In some embodiments, a fusion protein of the disclosure comprises, from N-terminus to C-terminus, an antigenic polypeptide, a transmembrane domain, an EPM, and an EIEIDo.

[0216] In some embodiments, a fusion protein of the disclosure comprises, from N-terminus to C-terminus, a signal peptide, an antigenic polypeptide, a transmembrane domain, and an EIEIDo. In some embodiments, a fusion protein of the disclosure comprises, from N-terminus to C-terminus, a signal peptide, an antigenic polypeptide, a transmembrane domain, an EPM, and an EIEIDo.

[0217] In some embodiments, a fusion protein of the disclosure comprises an antigenic polypeptide described herein and an EIEIDo described herein, wherein the antigenic polypeptide and the EIEIDo are linked. For example, in some embodiments, a fusion protein of the disclosure comprises an antigenic polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 1-5, and an EIEIDo comprising the amino acid sequence of any one of SEQ ID NOS: 6-120, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%. at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%. at least about 88%, at least about 89%, at least about 90%, at least about 91%. at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identitythereto. In some embodiments of the fusion proteins of the disclosure, the antigenic polypeptide and the EIEIDo are linked by a linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a glycine serine linker. In some embodiments, the linker comprises an amino acid sequence selected from Table 8C. In some embodiments, the linker comprises an amino acid sequence selected from Table 8D

[0218] In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence of SEQ ID NOs: 123-137, 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 identity7, 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.Antigenic Polypeptides

[0219] Also disclosed herein are exemplary antigenic polypeptides that can be used in the polypeptides or fusion proteins of the disclosure. By way of non-limiting examples, the antigenic polypeptides may comprise one or more polypeptides associated with viral or bacterial infection of a subject. For example, in some embodiments, the one or more antigenic polypeptides comprise beta coronavirus spike proteins (including SARS-CoV, MERS, SARS- CoV-2), antigenic polypeptides associated with Epstein-Barr viral infection (EBV) including gp42, gL, gH, gp350, BMRF2, or gB, antigenic polypeptides associated with influenza hemagglutinin (HA) including head and stem regions, neuraminidase (NA), antigenic polypeptides associated with Orthopoxvirus infection, antigenic polypeptides associated with flavivirus, antigenic polypeptides associated with chlamydia infection, antigenic polypeptides associated with staphylococcus aureus infection, antigenic polypeptides associated with infection from vibrio cholera, H. pylori, Streptococcus pneumoniae, meningococcal group B, or the like.

[0220] 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.

[0221] 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, aflavivirus, human metapneumo virus (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.

[0222] 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.

[0223] In some embodiments, the antigenic poly peptides 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 receptor-binding domain (RBD) of SARS-CoV spike protein.

[0224] 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 certain embodiments, the antigenic polypeptides described are derived from the receptor-binding domain (RBD) of MERS-CoV spike protein.

[0225] 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 the spike protein of SARS-CoV-2 or variant strain thereof.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] In some embodiments, the antigenic polypeptides described herein are derived from a protein of EBV.

[0234] In some embodiments, the antigenic poly peptides described herein are derived from a protein of a flavivirus. In some embodiments, the flavivirus is WNV or JEV. In certain embodiments, the antigenic polypeptides described herein are derived from NS 1 of WNV or JEV. In certain embodiments, the antigenic polypeptides described herein are derived from NS1 of WNV and JEV.

[0235] 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.

[0236] 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.

[0237] In some embodiments, an antigenic polypeptide of the disclosure is a protein associated with cancer. Examples of cancers from which an antigen polypeptide can be derived include, but are not limited to, breast cancer, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer, lymphoma, skin cancer, bladder cancer, prostate cancer, melanoma, or the like. In some embodiments, the antigenic polypeptide is derived from a prostate cancer associated protein. In certain embodiments, the prostate cancer associated protein is Six Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) or Prostate-specific membrane antigen (PSMA). In some embodiments, the antigenic polypeptide is derived from a melanoma associated protein. In certain embodiments, the melanoma associated protein is Tyrosinase or transmembrane phosphatase with tensin homology protein (TPTE).

[0238] Table 8A below provides exemplary antigens from which the antigenic polypeptides of the disclosure may be derived. In some embodiments, an antigenic polypeptide of the disclosure is derived from an antigen selected from Table 8A.Table 8A: Exemplary Antigens

[0239] In some embodiments, the antigenic polypeptides described herein comprises a MERS antigen comprising an amino acid sequence selected from Table 8B, or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%. at least about 82%, at least about 83%, at least about 84%. at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the MERS antigen comprises a signal peptide. In certain embodiments, the signal peptide comprises the sequence ATMIHSVFLLMFLLTPTESYWSHPQFEK (SEQ ID NO: 236), or an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%. at least about 80%, at least about 81%, at least about 82%, at least about 83%. at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0240] In some embodiments, the antigenic polypeptides described herein comprises a MERS antigen comprising the amino acid sequence of any one of SEQ ID NOS: 1-5, or an amino acid sequence having at least about 70%. at least about 71%. at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%. at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%. at least about 90%. at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.Table 8B: Exemplary MERS Polypeptide Amino Acid SequencesLinkers

[0241] 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 8C and Table 8D.

[0242] Table 8C provides exemplary amino acid sequences for flexible linkers.Table 8C. Exemplary flexible linker sequences

[0243] Table 8D provides exemplary amino acid sequences for rigid linkers.Table 8D. Exemplary rigid linker sequencesPolynucleotides

[0244] Disclosed herein are polynucleotides encoding one or more of the EIEIDOs described herein. Also disclosed herein are polynucleotides encoding a polypeptide comprising one or more of the EIEIDos described herein.

[0245] In some embodiments, the polynucleotides disclosed herein encode one or more EIEIDos which facilitate production of eVLPs. In some embodiments, the EIEIDos comprise: (1) an envelope protein of a virus, or a domain or fragment thereof; (2) a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof; (3) a lysosome- associated membrane protein 2 (LAMP2) polypeptide, or a domain or fragment thereof, or a binding partner thereof; (4) a polypeptide that binds to phosphatidylserine, or a domain or fragment thereof; (5) a platelet-derived growth factor receptor (PDGFR) polypeptide, or a domain or fragment thereof; or (6) a polypeptide capable of forming an amphipathic helix.

[0246] In some embodiments, the polynucleotides disclosed herein encode a polypeptide comprising (1) an envelope protein of a virus, or a domain or fragment thereof; (2) a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof; (3) a lysosome- associated membrane protein 2 (LAMP2) polypeptide, or a domain or fragment thereof, or a binding partner thereof; (4) a polypeptide that binds to phosphatidylserine, or a domain or fragment thereof; (5) a platelet-derived growth factor receptor (PDGFR) polypeptide, or a domain or fragment thereof; or (6) a polypeptide capable of forming an amphipathic helix.

[0247] Additionally, disclosed herein are polynucleotides encoding a fusion protein described herein. In some embodiments, the fusion protein comprises an antigenic polypeptide and an EIEIDo comprising (1) an envelope protein of a virus, or a domain or fragment thereof; (2) a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof; (3) a lysosome-associated membrane protein 2 (LAMP2) polypeptide, or a domain or fragment thereof, or a binding partner thereof; (4) a polypeptide that binds to phosphatidylserine, or a domain or fragment thereof; (5) a platelet-derived growth factor receptor (PDGFR) polypeptide, or a domain or fragment thereof; or (6) a polypeptide capable of forming an amphipathic helix.Also disclosed herein are polynucleotides encoding any one or more of the polypeptides described herein.

[0248] In some embodiments, the polynucleotides of the disclosure are administered to a subject to prevent and / or treat a disease or disorder. Also provided herein are polynucleotides that encode any one or more of the sequences, antigens, or polypeptides in Table 1, Table 2A. Table 2B, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8A, or Table SB

[0249] In some embodiments, a polynucleotide of the disclosure is or comprises DNA. In some embodiments, a polynucleotide of the disclosure is or comprises RNA. In some embodiments, a polynucleotide of the disclosure is or comprises mRNA.

[0250] In some embodiments, the polynucleotides encoding a fusion protein of the disclosure comprises a promoter. In some embodiments, the promoter comprises a ubiquitous promoter, an inducible promoter, a tissue-specific promoter and / or a lineage-specific promoter. In some embodiments, the ubiquitous promoter is selected from the group consisting of: a viral simian virus 40 (SV40) (e.g., early or late), a Rous sarcoma virus (RSV) LTR, an RSV promoter, a cytomegalovirus (CMV) immediate early promoter, a CMV promoter, a Moloney murine leukemia virus (MoMLV) LTR promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, HS, P7.S, and Pl 1 promoters from vaccinia virus, an elongation factor I-alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein S (HSP AS), heat shock protein 90 kDa beta, member 1 (HSP90B 1), heat shock protein 70 kDa (HSP70), P-kinesin (P-KIN). the human ROSA 26 locus, a Ubiquitin C promoter (UBC), a phosphoglycerate kinase-I (PGK) promoter, 3-phosphogly cerate kinase promoter, a cytomegalovirus enhancer, human P-actin (HBA) promoter, chicken P-actin (CBA) promoter, a CAG promoter, a CASI promoter, a CBH promoter, or any combination thereof.

[0251] In some embodiments, the polynucleotides encoding a fusion protein of the disclosure are operably linked to a tandem gene expression element (e g., an internal ribosomal entry site (IRES), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), porcine teschovirus 2A peptide (P2A) or Thosea asigna virus 2A peptide (T2A), poly A tails or any combination thereof. In some embodiments, the polynucleotides encoding a fusion protein comprise a transcript stabilization element (e.g., woodchuck hepatitis post-translational regulatory element (WPRE). bovine growth hormone polyadenylation (bGH-polyA) signalsequence, human grow th hormone polyadenylation (hGH-polyA) signal sequence, or any combination thereof.

[0252] In some embodiments, the polynucleotides encoding a fusion protein of the disclosure comprises a poly(A) tail. Inclusion of a 3‘ poly(A) tail in an mRNA sequence of the disclosure can contribute to the stabi 1 ity 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.

[0253] In some embodiments, the polynucleotides of the disclosure comprising 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.

[0254] In some embodiments, the polynucleotides of the disclosure comprise mRNA encoding a fusion protein of the disclosure. In some embodiments, the polynucleotides of the disclosure are formulated in a lipid nanoparticle (LNP). In some embodiments, the polynucleotides of the disclosure comprising mRNA are formulated in a lipid nanoparticle (LNP).

[0255] In some embodiments, the mRNA comprises a 5' untranslated region (UTR), a 3' UTR, and / or a cap. In some embodiments, the mRNA comprises a modification. In certain embodiments, the modification leads to a stabilization of the mRNA sequence. Therefore, also provided herein is a stabilized mRNA sequence comprising at least one coding region encoding a polypeptide or fusion protein described herein.

[0256] In some embodiments, the mRNA is essentially resistant to in vivo degradation (e.g., by an exo- or endo-nuclease). Such stabilization can be affected, for example, by a modified phosphate backbone of an mRNA. A backbone modification can be a modification in which phosphates of the backbone of the nucleotides contained in the mRNA are chemically modified. Nucleotides that can be used in this connection contain, for example, a phosphorothioate- modified phosphate backbone, such as at least one of the phosphate oxygens contained in the phosphate backbone being replaced by a sulfur atom. Stabilized mRNAs may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form. Such backbone modifications typically include, without implying any limitation, modificationsfrom the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5' -O-(l -thiophosphate)). Modifications to an mRNA may also include nucleotide analogues / modifications, e.g. sugar modifications or base modifications. A sugar modification can be a chemical modification of the sugar of the nucleotides of the mRNA compound comprising an mRNA sequence as defined herein. Furthermore, a base modification can be a chemical modification of the base moiety of the nucleotides of the mRNA compound comprising an mRNA sequence.

[0257] In some embodiments, the polynucleotides of the disclosure comprising mRNA comprise one or more modified nucleotides selected from the group consisting of pseudouridine, N-l-methyl-pseudouridine, 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3- methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5 -fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5- propynyl-cytidine, C5 -methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine. The mRNA can comprise a modified nucleotide in place of one or more uridines. The modified nucleoside can be selected from pseudouridine (vp), N 1 -methyl-pseudouridine (m h| / ). and 5-methyl-uridine (m5U). In some embodiments, the mRNA comprises a modified nucleotide in place of one or more uridines. In some embodiments, a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art.

[0258] In some embodiments, a polynucleotide encoding a fusion protein of the disclosure is 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 polynucleotide.

[0259] The polynucleotides of the disclosure can be included in an expression cassette for expression of a fusion protein encoded by the polynucleotides in a cell or an organism of interest. In some embodiments, the polynucleotides of the disclosure are codon-optimized for expression in a cell or an organism of interest.Vectors

[0260] Also disclosed herein are vectors comprising a polynucleotide of the disclosure. In some embodiments, the vector is a viral vector, a plasmid, a transposable element, a naked DNA vector, a lipid nanoparticle (LNP), or any combination thereof. In some embodiments, the viralvector is an AAV vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, a herpesvirus vector, a herpes simplex virus vector, a cytomegalovirus vector, a vaccinia virus vector, a MV A vector, a baculovirus vector, a vesicular stomatitis virus vector, a human papillomavirus vector, an avipox virus vector, a Sindbis virus vector, a VEE vector, a Measles virus vector, an influenza virus vector, a pox virus vector, a hepatitis B virus vector, an integration-deficient lentivirus (IDLV) vector, or any combination thereof.

[0261] In some embodiments, a vector of the disclosure comprises a viral vector that transport a polynucleotide encoding a fusion protein described herein into a cell without degradation and comprise a promoter yielding expression of the polynucleotide in the cell into which it is delivered.

[0262] In some embodiments, a vector of the disclosure is a lipid nanoparticle (LNP). In some embodiments, the LNP is associated with or encapsulates a polynucleotide (e.g., mRNA) encoding a fusion protein of the disclosure.

[0263] The term "lipid nanoparticle", also referred to as LNP, refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids. In some embodiments, such lipid nanoparticles comprise a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids, and polymer conjugated lipids (e.g.. a pegylated lipid). In some embodiments, the polynucleotide (e.g., mRNA), or a portion thereof, is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g., an adverse immune response. In some embodiments, the polynucleotide (e.g., mRNA) or a portion thereof is associated with the lipid nanoparticles. An LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. The term "lipid" refers to a group of organic compounds that are derivatives of fatty acids (e g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents.

[0264] The LNP can comprise one or more of an ionizable cationic lipid, a noncationic lipid (e.g., a neutral lipid), a sterol, and a PEG-modified lipid. The LNP can comprise 0.5-15 mol% PEG-modified lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 20-60. Mol% ionizable cationic lipid. The LNP can comprise 40-55 mol% ionizable cationic lipid, 5-15 Mol%neutral lipid, 35-45 mol% sterol, and 1-5 mol% PEG-modified lipid. In some embodiments, the RNA (e.g., mRNA) of the disclosure is formulated in a lipid nanoparticle (LNP). Lipid nanoparticles ty pically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest.

[0265] In some embodiments, the LNP comprises: 47 mol% ionizable cationic lipid, 11.5 mol% neutral lipid, 38.5 mol% sterol, and 3.0 mol% PEG-modified lipid; 48 mol% ionizable cationic lipid, 11 mol% neutral lipid, 38.5 mol% sterol, and 2.5 mol% PEG-modified lipid; 49 mol% ionizable cationic lipid, 10.5 mol% neutral lipid, 38.5 mol% sterol, and 2.0 mol% PEG- modified lipid; 50 mol% ionizable cationic lipid, 10 mol% neutral lipid, 38.5 mol% sterol, and 1.5 mol% PEG-modified lipid; or 51 mol% ionizable cationic lipid, 9.5 mol% neutral lipid, 38.5 mol% sterol, and 1.0 mol% PEG-modified lipid.

[0266] The ionizable cationic lipid can be heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6- (undecyloxy)hexyl)amino)octanoate. The neutral lipid can be 1,2 distearoyl-sn glycero-3 phosphocholine (DSPC). The sterol can be cholesterol. The PEG-modified lipid can be 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular yveight 2000 (PEG2000 DMG).

[0267] The wt / wt ratio of lipid to mRNA can be from about 1 : 100 to about 100: l(e.g., 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4. 1:1.5, 1:1.6, 1:1.7, 1:1.8. 1:1.9, 1:2, 1:2.5. 1:3. 1:4, 1:5, L6.L7. 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23,1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56. 1:57, 1:58, 1:59, 1:60, 1:61, 1 :62.1:63. 1:64. 1:65, 1 :66, 1:67.1:68. 1:69. 1:70, 1 :71, 1:72, 1:73.1:74.1:75. 1:76, 1:77, 1:78, 1 :79, 1:80, 1:81, 1:82, 1 :83, 1:84, 1:85, 1:86, 1:87, 1 :88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100 to 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1,2:1,2.5:1,3:1,41,5:1,6:1,71,8:1,9:1, 10:1, 11:1, 121, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1.30:1.31:1, 32:1,33:1.34:1.35:1.36:1.37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1.46:1.47:1.48:1.49:1.501, 51:1, 52:1, 53:1, 54:1, 551, 56:1, 57:1, 58:1, 591, 60:1, 61:1, 62:1, 63:1, 641, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1,84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1.98:1, 99:1, 100: l,or a number or a range between any of these values).

[0268] The LNP can comprise a cationic lipid. The cationic lipid may can be cation-isable, i.e., it becomes protonated as the pH is lowered below the pKa of the ionizable group of the lipid, but is progressively more neutral at higher pH values. When positively charged, the lipid is then able to associate with negatively charged nucleic acids. In some embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease. The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. In some embodiments, the LNP may comprise any further cationic or cation-isable lipid, i.e., any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N- dimethylammonium chloride (DOD AC); N-(2,3-dioleyloxy)propyl)-N,N,Ntrimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N- (2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N' ,N' dimethylaminoethane )-carbamoyl)cholesterol (DC-Chol), N-(l-(2,3-dioleoyloxy)propyl)N-2- (sperminecarboxamido )ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoy 1-3 -dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N- (l,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxy ethyl ammonium bromide (DMRIE).

[0269] Additionally, a number of commercial preparations of cationic lipids are available which can be used in embodiments provided herein. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1.2-dioleoyl-sn- 3phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(l-(2,3dioleyloxy)propyl)-N-(2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH DODAP, DODMA, DMDMA, l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinolenyloxy-N,Ndimethylaminopropane (DLenDMA).

[0270] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC). dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-lcarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-0-monomethyl PE, 16-O-dimethyl PE. 18-1- trans PE, lstearioyl-2-oleoylphosphatidyethanol amme (SOPE), and l,2-dielaidoyl-sn-glycero-3- Phophoethanol amine (transDOPE). In some embodiments, the neutral lipid is 1 ,2-distearoyl-sn- glycero-3 phosphocholine (DSPC).

[0271] In some embodiments, the cationic lipid is an amino lipid. Representative amino lipids include, but are not limited to, l,2-dilinoleyoxy-3-(dimethyl amino ) acetoxypropane (DLin- DAC), l,2-dilinoleyoxy-3morpholinopropane (DLinMA), l,2-dilinoleoyl-3- dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin-S- DMA). l-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3- trimethylaminopropane chloride salt (DLin-TMA.Cl),l,2-dihnoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3- (N,N-dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-di oleylamino )-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2-N,N-dimethylamino) ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl[l,3]-dioxolane (DLin-K-DMA).

[0272] In some embodiments, anon-cationic lipid comprises l,2-distearoyl-snglycero-3- phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cerophosphocholine (DMPC). l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoylsn- glycero-3- phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (Cl 6 Lyso PC), 1,2-dilinolenoy 1-sn- glycero-3-phosphocholine.l.2-diarachidonoyl-sn-gl ycero-3-phosphocholine. 1.2- didocosahexaenoy 1-sn-gl ycero-3-phosphocholine, 1,2-di phytanoy l-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine. 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 .2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac(l -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0273] In some embodiments, a PEG modified lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is DMG-PEG, PEG cDOMG (also referred to as PEG-DOMG), PEG-DSG and / or PEG-DPG. In some embodiments, a sterol comprises cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof.Cells

[0274] Disclosed herein is a cell that (1) expresses on its cell surface a fusion protein described herein or an antigenic polypeptide described herein and / or (2) generates an eVLPs 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. A cell receiving a polynucleotide encoding a fusion protein described herein may produce an eVLP of the disclosure, which is further described in detail herein. Such a cell may be referred to as a "host cell". In some embodiments, a host cell produces a fusion protein described herein. In some embodiments, a host cell is used for producing or maintaining nucleic acids, polynucleotides, or vectors of the disclosure. A host cell can be an in vitro, ex vivo, or in vivo host cell.

[0275] Populations of cells of the disclosure and cell cultures compnsing a host cell descnbed herein are also disclosed herein. In some embodiments, the cell is a prokaryotic cell, including, for example, a bacterial cell. Examples of prokary otic cells include, but are not limited to, cells of E. coli, Pseudomonas. Bacillus or Streptomyces. In some embodiments, the cell is a eukaryotic cell. Examples of eukaryotic cells include, but are not limited to, yeast cells such as cells of Saccharomyces yeast, animal cells such as CHO, RI.l, B-W and LM cells, African Green Monkey kidney cells (for example, COS 1, COS 7, BSCI, BSC40, and BMTIO), or human cells (such as human embry onic kidney cells, for instance, HEK293T, Expi293F. or HeLa cells). In some embodiments, the cell is a human cell.eVLPs

[0276] Also disclosed herein is an enveloped virus-like particle (eVLP) comprising a polypeptide or a fusion protein of the disclosure. In some embodiments, the enveloped virus-like particles (eVLP), generated using the polynucleotides, polypeptides, or fusion proteins described herein, displays on its surface an antigenic polypeptide described herein, or a portion thereof. In some embodiments, the enveloped virus-like particles (eVLP), generated using the polynucleotides, polypeptides, or fusion proteins described herein, displays on its surface MHC class I molecules and an antigenic polypeptide described herein, or a portion thereof.

[0277] 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 EIEIDos described herein. In some embodiments, the size of the eVLPs produced using the methods of the disclosure ranges from about 10 nm to about 1000 nm. In some embodiments, the size of the eVLPs ranges from about 10 nm to about 150 nm. In certain embodiments, the size of an eVLP is about lOnm. about 20 nm. about 25 nm. about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about lOOnm, about 125nm, about 150nm, about 175nm, about 200nm, about 300nm, about 400nm, about 500nm, about 600nm, about 700nm, about 800nm, about 900nm, or about lOOOnm.Methods and Uses

[0278] Disclosed herein are methods of treating or preventing a disease or disorder, or a symptom thereof, in a subject in need thereof.

[0279] Also disclosed herein are methods of vaccinating a subject in need thereof, wherein the subject has been diagnosed with an infection or is at risk of being infected by a pathogen. In some embodiments, the pathogen is a virus, a bacterium, or a parasite. In certain embodiments, the pathogen is a virus. In certain embodiments, the pathogen is a bacterium. In certain embodiments, the pathogen is a parasite.

[0280] In some embodiments, the methods of treating, preventing, or vaccinating comprise administering to the subject in need thereof an effective amount of a polynucleotide of thedisclosure, a vector of the disclosure, an eVLP of the disclosure, or a cell of the disclosure. In some embodiments, the methods comprise administering to the subject in need thereof an effective amount of a polynucleotide encoding a fusion protein comprising an antigenic polypeptide and an EIEIDo comprising an amino acid sequence of SEQ ID NOS: 6-207 or 218- 232, or an ammo acid sequence having at least 70% sequence identity thereto. In some embodiments, the methods comprise administering to the subject in need thereof at least a first dose of an effective amount of a polynucleotide of the disclosure, a vector of the disclosure, an eVLP of the disclosure, or a cell of the disclosure.

[0281] Also disclosed herein are uses of a polynucleotide of the disclosure, a polypeptide of the disclosure, a fusion protein of the disclosure, a vector of the disclosure, an eVLP of the disclosure, or a cell of the disclosure for the manufacture of a medicament or vaccine composition. Additionally, disclosed herein uses of a polynucleotide of the disclosure, a polypeptide of the disclosure, a fusion protein of the disclosure, a vector of the disclosure, an eVLP of the disclosure, or a cell of the disclosure for the manufacture of a medicament or vaccine composition for the prevention or treatment of a disease or disorder. In some embodiments, the medicament or vaccine composition comprises polynucleotides of the disclosure. In some embodiments, the medicament or vaccine composition comprises vectors of the disclosure. In some embodiments, eVLPs are isolated from supernatant of cells in culture which were transfected with polynucleotides or vectors of the disclosure, and used in a medicament or vaccine composition. In some embodiments, the medicament or vaccine composition comprises cells which were transfected with polynucleotides or vectors of the disclosure.

[0282] Also disclosed herein are the following non-limiting uses for the polynucleotides of the disclosure, the polypeptides of the disclosure, the fusion proteins of the disclosure, the vectors of the disclosure, the eVLPs of the disclosure, or the cells of the disclosure are contemplated: (1) for treatment or prevention of a disease or disorder, (2) for vaccination. (3) for inducing antigenspecific antibodies, wherein the antibodies bind to an antigenic polypeptide of the disclosure, or a portion thereof, (4) for inducing an immune response against a virus, (5) for inducing an immune response against a bacterium, (6) for in inducing an immune response against a parasite, and (7) for inducing an immune response against a cancer.

[0283] In some embodiments of the methods or uses of the disclosure, the disease or disorder is an infection. In some embodiments, the infection is a viral infection. Examples of viralinfections 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. In some embodiments, the infection is an infection by a parasite.

[0284] In some embodiments, the infection comprises an infection by Acute Flaccid Myelitis (AFM), Anaplasmosis. Anthrax, Babesiosis, Botulism, Brucellosis, Campy lobacteriosis, Carbapenem-resistant Infection, Chancroid, Chikungunya Virus Infection, Chlamydia.Ciguatera, Difficile Infection, Perfringens, Coccidioidomycosis fungal infection, coronavirus infection, Covid-19 (SARS-Co V-2), Creutzfeldt-Jacob Disease / transmissible spongiform encephalopathy, Cryptosporidiosis (Crypto), Cyclosporiasis, Dengue 1.2,3 or 4, Diphtheria, E. coli infection / Shigatoxin-producing (STEC), Eastern Equine Encephalitis, Hemorrhagic Fever (Ebola), Ehrlichiosis, Encephalitis, Arboviral or parainfectious, Non-Polio Enterovirus, D68 Enteroviru (EV -D68), Giardiasis, Glanders, Gonococcal Infection, Granuloma inguinale, Haemophilus Influenza disease Type B (Hib or H-flu), Hantavirus Pulmonary7Syndrome (HPS), Hemolytic Uremic Syndrome (HUS), Hepatitis A (Hep A), Hepatitis B (Hep B), Hepatitis C (Hep C), Hepatitis D (Hep D). Hepatitis E (Hep E), Herpes. Herpes Zoster (Shingles), Histoplasmosis infection. Human Immunodeficiency Virus / AIDS (HIV / AIDS), Human Papillomavirus (HPV), Influenza (Flu), Legionellosis (Legionnaires Disease), Leprosy (Hansens Disease), Leptospirosis, Listeriosis (Listeria), Lyme Disease, Lymphogranuloma venereum infection (LGV). Malaria, Measles, Melioidosis, Meningitis (Viral), Meningococcal Disease (Meningitis (Bacterial)), MERS, Mumps, Norovirus, Pediculosis, Pelvic Inflammatory Disease (PID), Pertussis (Whooping Cough), Plague (Bubonic, Septicemic, Pneumonic), Pneumococcal Disease (Pneumonia), Poliomyelitis (Polio), Powassan, Psittacosis, Pthiriasis, Pustular Rash diseases (Small pox, monkeypox, cowpox), Q-Fever, Rabies, Rickettsiosis. (Rocky Mountain Spotted Fever), Rubella (German Measles). Salmonellosis gastroententis(Salmonella). Scabies, Scombroid, Sepsis, Severe Acute Respiratory Syndrome (SARS), Shigellosis gastroenteritis (Shigella), Smallpox, Staphyloccal Infection Methicillin-resistant (MRS A), Staphylococcal Food Poisoning Enterotoxin B Poisoning (Staph Food Poisoning), Saphylococcal Infection Vancomycin Intermediate (VISA), Staphylococcal Infection Vancomycin Resistant (VRSA), Streptococcal Disease Group A (invasive) (Strep A (invasive). Streptococcal Disease, Group B(Strep-B), Streptococcal Toxic-Shock Syndrome STSS Toxic Shock, Syphilis (primary, secondary, early latent, late latent, congenital), Tetanus Infection, Trichomoniasis, Trichonosis Infection, Tuberculosis (TB), Tuberculosis Latent (LTBI), Tularemia, Typhoid Fever Group D, Vaginosis, Varicella (Chickenpox), Vibrio cholerae (Cholera), Vibriosis (Vibrio), Ebola Virus Hemorrhagic Fever, Lasa Virus Hemorrhagic Fever, Marburg Virus Hemorrhagic Fever. West Nile Virus, Yellow Fever, Yersenia, or Zika Virus Infection.

[0285] In some embodiments of the methods or uses of the disclosure, the disease or disorder is cancer.

[0286] In some embodiments, the cancer is a hematologic cancer chosen from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, Hodgkin's Disease, non-Hodgkin lymphoma, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell- follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle celllymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm. Waldenstrom macroglobulinemia. or preleukemia.

[0287] In some embodiments, the cancer comprises prostate cancer, colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma. Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

[0288] In some embodiments of the methods of the disclosure, the administration is in the context of treatment, and used for the mitigation of an already existing infection and optionally facilitates antibody-mediated clearance of the infection.

[0289] In some embodiments, the methods of treating, preventing, or vaccinating comprise administering to the subject an additional dose of an effective amount of a polynucleotide of the disclosure, a vector of the disclosure, an eVLP of the disclosure, or a cell of the disclosure. In some embodiments, administering to the subject an additional dose comprises administering to the subject at least two additional doses, at least three additional doses, at least four additional doses, or at least five additional doses of an effective amount of a polynucleotide of the disclosure, a vector of the disclosure, an eVLP of the disclosure, or a cell of the disclosure. In certain embodiments, administering to the subject an additional dose of an effective amount of a polynucleotide of the disclosure comprises administering to the subject a second dose of an effective amount of a polynucleotide of the disclosure.

[0290] 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. In some embodiments, the route of administration may include injection, inhalation or insulation or by oral, parenteral or rectal administration. In some embodiments, the route of administration may use traditional syringes and needleless injection devices. Suitable routes of administration include, but are not limited to, parenteral delivery, such as intramuscular, intradermal, subcutaneous, intramedullary injections, as well as, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the effective amount of any one of the polynucleotides of the disclosure herein described can be formulated in aqueous solutions, optionally in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. In some embodiments, administering can comprise aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery', ocular delivery, local delivery, topical delivery', intracistemal delivery', intraperitoneal delivery, oral delivery', intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof.

[0291] In some embodiments, the subject in need thereof is treated with a polynucleotide described herein, a vector described herein, an eVLP described herein, or a cell described herein. In some embodiments, the subject is a mammal. Non-limiting examples of mammals include, a human, a non-human primate, a rodent, a dog. a cat, a rabbit, a cow, a horse, a goat, a sheep, a llama, an alpaca, 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 is a bird, such as a chicken, a duck, a pheasant, a turkey, or a goose. In some embodiments, the subject in need thereof is not a human subject.

[0292] 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 some embodiments, the subject being treated in accordance with the methods described herein has been diagnosed with a viral infection or is at risk of being infected by a virus. In some embodiments, the subject being treated in accordance with the methods described herein has been diagnosed with a bacterial infection or is at risk of being infected by a 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. In some embodiments, the subject being treated in accordance with the methods described herein has been diagnosed with or is at risk of having cancer.

[0293] In some embodiments, the methods of treating, preventing, or vaccinating comprise administering to the subject in need thereof an effective amount of a polynucleotide described herein within an LNP. In some embodiments, the methods of treating or preventing a disease or disorder in a subject in need thereof comprise administering to the subject in need thereof an additional dose of a polynucleotide described herein within an LNP.

[0294] 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.EXEMPLARY EMBODIMENTS

[0295] Embodiment 1-1. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises:(a) an immunogenic polypeptide; and(b) a polypeptide comprising an ESCRT-independent eVLP inducing domain.

[0296] Embodiment 1-2. The polynucleotide of embodiment 1-1, wherein the fusion protein comprises:(a) an immunogenic polypeptide; and(b) an envelope protein of a coronavirus.

[0297] Embodiment 1-3. The polynucleotide of embodiment 1-2, wherein the envelope protein of a coronavirus comprises a cytoplasmic tail of the envelope protein.

[0298] Embodiment 1-4. The polynucleotide of any embodiment of embodiments 1-2 to 1-3, wherein the envelope protein is selected from a group consisting of: avian infectious bronchitis virus (AIBV) mouse hepatitis virus (MHV), and transmissible gastroenteritis virus (TGEV).

[0299] Embodiment 1-5. The polynucleotide of any embodiment of embodiments 1-2 to 1-4, wherein the envelope protein comprises any amino acid sequence of SEQ ID NOS: 6-9, or an amino acid sequence having 70% sequence identity thereto.

[0300] Embodiment 1-6. The polynucleotide of embodiment 1-1, wherein the fusion protein comprises:(a) an immunogenic polypeptide; and one of:(i) a tetraspanin polypeptide, a domain or fragment thereof; or(ii) a binding partner of a tetraspanin polypeptide.

[0301] Embodiment 1-7. The polynucleotide of embodiment 1-6, wherein the tetraspanin polypeptide is selected from the group consisting of: CD9, CD63, CD81. CD82, CD37, CD53. CD151, TSP-1, TSP-2, TSP-3, TSP-4, TSP-5, and TSP-6.

[0302] Embodiment 1-8. The polynucleotide of any one of embodiments 1-6 to 1-7, wherein the tetraspanin polypeptide comprises any amino acid sequence of SEQ ID NOS: 10-13, or an amino acid sequence having 70% sequence identity thereto.

[0303] Embodiment 1-9. The polynucleotide of any one of embodiments 1-6 to 1-8, wherein the binding partner of the tetraspanin polyptide comprises a human cytomegalovirus (EICMV) gM cytoplasmic tail.

[0304] Embodiment 1-10. The polynucleotide of embodiment 1-9, wherein the HCMV cytoplasmic tail comprises an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having 70% sequence identity thereto.

[0305] Embodiment 1-11. The polynucleotide of embodiment 1-6, wherein the tetraspanin polypeptide comprises a homologous tetraspanin polypeptide from a non-human vertebrate species or an invertebrate species.

[0306] Embodiment 1-12. The polynucleotide of embodiment 1-11, wherein the non-human vertebrate species or invertebrate species is selected from the group consisting of: Atlantic canary. Florida worm lizard. Adelie penguin, Common toad, Orbiculate cardinalfish. Aardvark, Diamondback terrapin, MacQueen's bustard. Green anole, and Common wombat.

[0307] Embodiment 1-13. The polynucleotide of embodiment 1-1, wherein the fusion protein comprises:(a) an immunogenic polypeptide; and (b) one of the following:(i) a lysosome-associated membrane protein 2 (LAMP 2) polypeptide, domain or fragment thereof; or(ii) a binding partner of the LAMP 2 peptide

[0308] Embodiment 1-14. The polynucleotide of embodiment 1-13, wherein the LAMP 2 polypeptide comprises a LAMP2A polypeptide or a LAMP2B polypeptide.

[0309] Embodiment 1-15. The polynucleotide of embodiment 1-13 or 1-14, wherein the LAMP 2 polypeptide comprises an amino acid sequence of SEQ ID NOS: 15 or 16, or an amino acid sequence having 70% sequence identity thereto.

[0310] Embodiment 1-16. The polynucleotide of embodiment 1-13. wherein the binding partner of the LAMP 2 polypeptide comprises a motif of KFERQ (SEQ ID NO: 17).

[0311] Embodiment 1-17. The polynucleotide of embodiment 1-13 or 1-1 , wherein the binding partner of the LAMP 2 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 18-20.

[0312] Embodiment 1-18. The polynucleotide of embodiment 1-1, wherein the fusion protein comprises:(a) an immunogenic polypeptide; and(b) a phosphatidylserine-binding polypeptide, domain, or fragment thereof.

[0313] Embodiment 1-19. The polynucleotide of embodiment 1-18, wherein the polypeptide, domain or fragment thereof that binds to phosphatidylserine is derived from lactadherin or comprises a lactadherin polypeptide.

[0314] Embodiment 1-20. The polynucleotide of embodiment 1-19, wherein the lactadherin polypeptide comprises a ClC2 domain.

[0315] Embodiment 1-21. The polynucleotide of embodiment 1-20, wherein the C1C2 domain comprises an amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 70% sequence identity thereto.

[0316] Embodiment 1-22. A polynucleotide of embodiment 1-1 encoding a fusion protein, wherein the fusion protein comprises:(a) an immunogenic polypeptide; and(b) a platelet-derived growth factor receptor (PDGFR) polypeptide, domain, or fragment thereof.

[0317] Embodiment 1-23. The polynucleotide of embodiment 1-22, wherein the PDGFR polypeptide, domain or fragment thereof comprises a homologous PDGFR polypeptide from a non-human vertebrate species or an invertebrate species.

[0318] Embodiment 1-24. The polynucleotide of embodiment 1-23, wherein the homologous PDGFR polypeptide is selected from the group consisting of: Spanish mole, brushtail possum, western clawed frog, Chinese alligator, and the common sole fish.

[0319] Embodiment 1-25. The polynucleotide of any one of embodiments 1-22 to 1-24, wherein the PDGFR polypeptide comprises a transmembrane domain.

[0320] Embodiment 1-26. The polynucleotide of any one of embodiments 1-22 to 1-25. wherein the PDGFR polypeptide comprises an amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 70% sequence identity thereto.

[0321] Embodiment 1-27. The polynucleotide of embodiment 1-1, wherein the fusion protein comprises:(a) an immunogenic polypeptide; and(b) a polypeptide comprising one or more amphipathic helices.

[0322] Embodiment 1-28. The polynucleotide of embodiment 1-27 wherein the one or more amphipathic helices are capable of inducing membrane fission by increasing membrane curvature.

[0323] Embodiment 1-29. The polynucleotide of embodiment 1-27 or 1-28, wherein the polypeptide comprising one or more amphipathic helices is isolated or derived from the group consisting of: influenza A M2 protein, EcMurG protein, CALM protein, or Drosophila amphiphay sin protein.

[0324] Embodiment 1-30. The polynucleotide of any one of embodiments 1-27 to 1-29. wherein the polypeptide comprises an amphipathic helix comprising an amino acid sequence selected from a group consisting of: SEQ ID NOS: 23-40, or an amino acid sequence having at least 70% sequence identity thereto.

[0325] Embodiment 1-31. The polynucleotide of any one of embodiments 1-27 to 1-29. wherein the polypeptide comprises two or more amphipathic helices.

[0326] Embodiment 1-32. The polynucleotide of embodiment 1-31, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 41-120, or an amino acid sequence having at least 70% sequence identity thereto.

[0327] Embodiment 1-33. The polynucleotide of embodiment 1-31 or 1-32, wherein each of the two or more amphipathic helices are coupled by a linker.

[0328] Embodiment 1-34. The polynucleotide of any one of embodiments 1-1 to 1-33 wherein the immunogenic polypeptide is derived from a viral protein.

[0329] Embodiment 1-35. The polynucleotide of embodiment 1-34. wherein the viral protein is an EBV protein, MERS protein, influenza protein, flavivirus protein, orthopoxvirus protein, or coronavirus protein.

[0330] Embodiment 1-36. The polynucleotide of any one of embodiments 1-1 to 1-33, wherein the immunogenic polypeptide is derived from a non-viral protein.

[0331] Embodiment 1-37. The polynucleotide of embodiment 1-36, wherein the immunogenic polypeptide is derived from a bacterial protein.

[0332] Embodiment 1-38. The polynucleotide of embodiment 1-36, wherein the immunogenic polypeptide is derived from a cancer associated protein.

[0333] Embodiment 1-39. The polynucleotide of any one of embodiments 1-1 to 1-38, wherein the polynucleotide comprises RNA.

[0334] Embodiment 1-40. The polynucleotide of any one of embodiments 1-1 to 1-38, wherein the polynucleotide comprises mRNA.

[0335] Embodiment 1-41. The polynucleotide of any one of embodiments 1-1 to 1-38, wherein the polynucleotide comprises DNA.

[0336] Embodiment 1-42. A fusion protein encoded by any one of the polynucleotides of embodiments 1-1 to 1-41.

[0337] Embodiment 1-43. A fusion protein, wherein the fusion protein has an amino acid sequence comprising any one of the sequences of SEQ ID NOS: 1-120. or a sequence having at least 70% sequence identity thereto.

[0338] Embodiment 1-44. A cell expressing on its surface the fusion protein of embodiment I- 42 or 1-43.

[0339] Embodiment 1-45. An enveloped virus-like particle (eVLP) comprising the polynucleotide of any one of embodiments 1-1 to 1-41 or the fusion protein of embodiment 1-42 or 1-43.

[0340] Embodiment 1-46. An enveloped virus-like particle (eVLP) displaying on its surface the immunogenic polypeptide, or part of the immunogenic polypeptide, of the fusion protein encoded by the polynucleotide of any one of embodiments 1-1 to 1-41.

[0341] Embodiment 1-47. The eVLP of embodiment 1-45 or 1-46, wherein the average size / diameter of the eVLP ranges from 20 nm to 200 nm.

[0342] Embodiment 1-48. A vector comprising the polynucleotide of any one of embodiments1-1 to 1-41.

[0343] Embodiment 1-49. The vector of embodiment 1-48, wherein the vector is a viral vector.

[0344] Embodiment 1-50. The vector of embodiment 1-48, wherein the vector is a non-viral vector.

[0345] Embodiment 1-51. The vector of embodiment 1-50, wherein the non-viral vector is a plasmid.

[0346] Embodiment 1-52. The vector of embodiment 1-50, wherein the non-viral vector is a lipid nanoparticle (LNP).

[0347] Embodiment 1-53. The vector of embodiment 1-50, wherein the non-viral vector is a lipid nanoparticle (LNP) and the polynucleotide comprises mRNA.

[0348] Embodiment 1-54. 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 1-1 to 1-41, the fusion protein of embodiment 1-42 or 1-43, the eVLP of any one of embodiments 1-45 to 1-47, or the vector of any one of embodiments1-48 to 1-53.

[0349] Embodiment 1-55. The method of embodiment 1-54, wherein the subject is a human subject.

[0350] Embodiment II- 1. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises:(a) an antigenic polypeptide; and(b) a polypeptide comprising an ESCRT-independent eVLP inducing domain (EIEIDo).

[0351] Embodiment II-2. The polynucleotide of embodiment II- 1 , wherein the EIEIDo comprises:(a) an envelope protein of a virus, or a domain or fragment thereof;(b) a tetraspanin polypeptide, or a domain or fragment thereof;(c) a binding partner of a tetraspanin polypeptide;(d) a Lysosome- Associated Membrane Protein 2 (LAMP2) polypeptide, or a domain or fragment thereof;(e) a binding partner of a LAMP2 polypeptide;(1) a phosphatidylserine-binding polypeptide, or a domain or fragment thereof;(g) a Platelet-Derived Growth Factor Receptor (PDGFR) polypeptide, or a domain or fragment thereof; or(h) a polypeptide comprising an amphipathic helix.

[0352] Embodiment II-3. The polynucleotide of embodiment II-2, wherein the EIEIDo comprises an envelope protein of a virus, or a fragment thereof.

[0353] Embodiment II-4. The polynucleotide of embodiment II-3, wherein the EIEIDo comprises the cytoplasmic tail of the envelope protein.

[0354] Embodiment II-5. The polynucleotide of any one of embodiments II-2 to II-4, wherein the virus is a coronavirus.

[0355] Embodiment II-6. The polynucleotide of any one of embodiments II-2 to II-5, wherein the coronavirus is a Gammacoronavirus, a Betacoronavirus, or an Alphacoronavirus.

[0356] Embodiment II-7. The polynucleotide of any one of embodiments II-2 to II-6, wherein the virus is selected from a group consisting of: Severe Acute Respiratory’ Syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, avian infectious bronchitis virus (AIBV), mouse hepatitis virus (MHV), and transmissible gastroenteritis virus (TGEV).

[0357] Embodiment II-8. The polynucleotide of any embodiment of embodiments II-2 to II-7, wherein the envelope protein comprises the amino acid sequence of any one of SEQ ID NOS: 6- 9, or an amino acid sequence having at least 70% sequence identity thereto.

[0358] Embodiment II-9. The polynucleotide of embodiment II-2, wherein the EIEIDo comprises a tetraspamn polypeptide, or a domain or fragment thereof.

[0359] Embodiment II- 10. The polynucleotide of embodiment II-9, wherein the tetraspanin polypeptide comprises a human tetraspanin polypeptide or a homologous tetraspanin polypeptide from a non-human vertebrate species or an invertebrate species.

[0360] Embodiment II- 11. The polynucleotide of embodiment II- 10, wherein the tetraspanin polypeptide comprises a human tetraspanin polypeptide.

[0361] Embodiment 11-12. The polynucleotide of embodiment II- 10, wherein the tetraspanin polypeptide comprises a tetraspanin polypeptide from anon-human vertebrate species or an invertebrate species.

[0362] Embodiment 11-13. The polynucleotide of embodiment 11-12, wherein the non-human vertebrate species or invertebrate species is selected from the group consisting of: Spanish mole, brushtail possum, western clawed frog, Chinese alligator, common sole fish, Atlantic canary, Florida worm lizard, Adelie penguin, Common toad, Orbiculate cardinalfish. Aardvark, Diamondback terrapin, MacQueen's bustard, Green anole, Common wombat, Budorcas taxicolor, Patagioenas fasciata, Maylandia zebra, Hypanus sabinus, Echinops telfairi. Pan troglodytes, Lagopus leucura, Thamnophis elegans, Omithorhynchus anatinus, Etheostoma spectabile, Carassius Carassius, Pleuronectes platessa, Monodon monoceros, Motacilla alba alba, Syngnathus typhle. Xiphophorus couchianus, Lonchura striata, Meriones unguiculatus. Vicugna pacos, Podarcis muralis, Sinocyclocheilus anshuiensis, Sorex fumeus, Ursus arctos, Myotis davidii, Bos taurus, Gopherus flavomarginatus, Orycteropus afer afer, Phasianus colchicum, Actinia equina, Orbicella faveolata, Daphnia carinata, Microtus oregoni, Petaurus breviceps papuanus, Gallus gallus, Myotis lucifugus, Malaclemys terrapin pileata, Bufo bufo , Zonotrichia leucophrys gambehi, Anolis sagrei. Leopardus geoffroyi. Manacus candei. and Sapajus apella.

[0363] Embodiment 11-14. The polynucleotide of any one of embodiments II-9 to 11-13, wherein the tetraspanin polypeptide is derived from a protein selected from the group consisting of: CD9, CD63, CD81, CD82. CD37, CD53, CD151, CD231, tetraspanin (TSP)-l. TSP-2. TSP- 3, TSP-4, TSP-5, TSP-6, TSP-9, TSP-11, and proteins encoded by genes TSPAN1-TSPAN33.

[0364] Embodiment 11-15. The polynucleotide of embodiment 11-14, wherein the tetraspanin polypeptide is derived from CD9.

[0365] Embodiment 11-16. The poly nucleotide of embodiment 11-15, wherein the tetraspanin polypeptide is derived from human CD9 or CD9 from Aardvark, Diamondback terrapin, MacQueen's bustard. Green anole. Common wombat. Budorcas taxicolor, Patagioenas fasciata, Maylandia zebra. Hypanus sabinus, Echinops telfairi, Pan troglodytes, Lagopus leucura, Thamnophis elegans, Omithorhynchus anatinus, Etheostoma spectabile, Carassius Carassius, Pleuronectes platessa, Monodon monoceros, Motacilla alba alba, or Syngnathus typhle.

[0366] Embodiment II- 17. The polynucleotide of embodiment II- 15, wherein the tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 10, 138-152, and 223-227, or an amino acid sequence having at least 70% sequence identity thereto.

[0367] Embodiment II- 18. The poly nucleotide of embodiment 11-14, wherein the tetraspanin polypeptide is derived from CD63.

[0368] Embodiment II- 19. The polynucleotide of embodiment II- 18, wherein the tetraspanin polypeptide is derived from human CD63 or CD63 from Actinia equina, Orbicella faveolata, Daphnia carinata, Microtus oregoni, Petaurus breviceps papuanus, Gallus gallus, Myotis lucifugus, Malaclemys terrapin pileata, Bufo bufo, Zonotrichia leucophrys gambelii, Sorex fumeus, Anolis sagrei. Leopardus geoffroyi. Manacus candei. or Sapajus apella.

[0369] Embodiment 11-20. The polynucleotide of embodiment II- 18 or 11-19, wherein the tetraspanin polypeptide comprises a modification in an endosome-targeting signal in CD63.

[0370] Embodiment 11-21. The polynucleotide of embodiment 11-20, wherein the endosometargeting signal comprises a modification of a tyrosine (Y) in the endosome-targeting signal in CD63, wherein the modification is an amino acid substitution.

[0371] Embodiment 11-22. The polynucleotide of embodiment 11-20 or 11-21, comprising a modification of a glutamate (E) in the endosome-targeting signal in CD63.

[0372] Embodiment 11-23. The polynucleotide of any one of embodiments 11-20 to 11-22, comprising a modification of a valine (V) in the endosome-targeting signal in CD63.

[0373] Embodiment 11-24. The polynucleotide of any one of embodiments 11-20 to 11-23, comprising a modification of a methionine (M) in the endosome-targeting signal in CD63.

[0374] Embodiment 11-25. The polynucleotide of any one of embodiments 11-20 to 11-24, comprising an amino acid substitution of tyrosine (Y) to alanine (A) in the endosome-targeting signal in CD63.

[0375] Embodiment 11-26. The polynucleotide of any one of embodiments 11-18 to 11-20, comprising a modification at a position corresponding to position 235, 236, 237, and / or 238 relative to SEQ ID NO: 11.

[0376] Embodiment 11-27. The polynucleotide of embodiment 11-26, comprising a modification at a position corresponding to position 235 relative to SEQ ID NO: 11.

[0377] Embodiment 11-28. The polynucleotide of embodiment 11-27, wherein the modification is an amino acid substitution of tyrosine (Y) to alanine (A).

[0378] Embodiment 11-29. The polynucleotide of any one of embodiments 11-18 to 11-28, wherein the tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 11 and 168-183, or an amino acid sequence having at least 70% sequence identity thereto.

[0379] Embodiment 11-30. The polynucleotide of embodiment 11-14, wherein the tetraspanin polypeptide is derived from CD81.

[0380] Embodiment II-31. The polynucleotide of embodiment 11-30, wherein the tetraspanin polypeptide is derived from human CD81 or CD81 from Atlantic canary, Florida worm lizard, Adelie penguin, Common toad, Orbiculate cardinalfish, Xiphophorus couchianus, Lonchura striata, Meriones unguiculatus, Vicugna pacos, Hypanus sabinus, Podarcis muralis, Sinocyclocheilus anshuiensis, Sorex fumeus, Ursus arctos, Myotis davidii, Patagioenas fasciata, Bos taurus. Gopherus flavomarginatus, Orycteropus afer afer. or Phasianus colchicus.

[0381] Embodiment 11-32. The polynucleotide of embodiment 11-30 or 11-31 , wherein the tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 12, 153- 167, and 218-222, or an amino acid sequence having at least 70% sequence identity thereto.

[0382] Embodiment 11-33. The polynucleotide of any one of embodiments II-9 to 11-32, wherein the EIEIDo comprises a chimeric tetraspanin polypeptide, wherein the chimeric tetraspanin polypeptide comprises a first tetraspanin polypeptide, wherein a domain in that first tetraspanin polypeptide is substituted with a domain from a second tetraspanin polypeptide.

[0383] Embodiment 11-34. The polynucleotide of embodiment 11-33, wherein the first and second tetraspanin polypeptides are different.

[0384] Embodiment 11-35. The polynucleotide of embodiment 11-33 or 11-34, wherein the first and second tetraspanin polypeptides are independently selected from the group consisting of: CD9, CD63, CD81, CD82, CD37, CD53, CD151, CD231, tetraspanin (TSP)-l, TSP-2, TSP-3. TSP-4. TSP-5, TSP-6, TSP-9, TSP-11, proteins encoded by genes TSPAN1-TSPAN33, and domains or fragments thereof.

[0385] Embodiment 11-36. The polynucleotide of any one of embodiments 11-33 to 11-35, wherein the chimeric tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 184-205, or an amino acid sequence having at least 70% sequence identity thereto.

[0386] Embodiment 11-37. The polynucleotide of any one of embodiments 11-9 to 11-35, wherein the EIEIDo further comprises a PDZ1 domain.

[0387] Embodiment 11-38. The polynucleotide of embodiment 11-37, wherein the EIEIDo comprises the amino acid sequence of SEQ ID NO: 206 or 207. or an amino acid sequence having at least 70% sequence identity thereto.

[0388] Embodiment 11-39. The polynucleotide of embodiment II-2, wherein the EIEIDo comprises a binding partner of a tetraspanin polypeptide, or a domain or fragment thereof.

[0389] Embodiment 11-40. The poly nucleotide of embodiment 11-39, wherein the tetraspanin is CD63.

[0390] Embodiment 11-41. The polynucleotide of embodiment 11-39 or 11-40, wherein the binding partner is a human cytomegalovirus (HCMV) protein.

[0391] Embodiment 11-42. The polynucleotide of embodiment 11-41, wherein the HCMV protein is glycoprotein M (gM).

[0392] Embodiment 11-43. The polynucleotide of embodiment 11-42. wherein the binding partner comprises the cytoplasmic tail of HCMV gM.

[0393] Embodiment 11-44. The polynucleotide of embodiment 11-43, wherein the cytoplasmic tail of HCMV gM comprises SEQ ID NO: 13 or an amino acid sequence having at least 70% sequence identity’ thereto.

[0394] Embodiment 11-45. The polynucleotide of embodiment II-2, wherein the EIEIDo comprises a Lysosome- Associated Membrane Protein 2 (LAMP2) polypeptide, or a domain or fragment thereof.

[0395] Embodiment 11-46. The polynucleotide of embodiment 11-45, wherein the EIEIDo comprises a LAMP2A polypeptide, a LAMP2B polypeptide, or a LAMP2C polypeptide.

[0396] Embodiment 11-47. The polynucleotide of embodiment 11-45 or 11-46, wherein the LAMP2 polypeptide comprises a human LAMP2 polypeptide or a homologous LAMP2 polypeptide from a non-human vertebrate species or an invertebrate species.

[0397] Embodiment 11-48. The polynucleotide of embodiment 11-47, wherein the LAMP2 polypeptide comprises a human LAMP2 polypeptide.

[0398] Embodiment 11-49. The polynucleotide of embodiment 11-47, wherein the LAMP2 polypeptide comprises a LAMP2 polypeptide from a non-human vertebrate species or an invertebrate species.

[0399] Embodiment 11-50. The polynucleotide of any one of embodiments 11-45 to 11-49, wherein the LAMP2 polypeptide comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence having at least 70% sequence identity thereto.

[0400] Embodiment II-51. The polynucleotide of embodiment II-2, wherein the EIEIDo comprises a binding partner of a LAMP2 polypeptide, or a domain or fragment thereof.

[0401] Embodiment 11-52. The polynucleotide of embodiment 11-51, wherein the binding partner comprises a motif, wherein the motif is configured to help a LAMP2 polypeptide redirect proteins to eVLPs.

[0402] Embodiment 11-53. The poly nucleotide of embodiment 11-52, wherein the motif comprises the amino acid sequence KFERQ (SEQ ID NO: 17).

[0403] Embodiment 11-54. The polynucleotide of any one of embodiments II-51 to 11-53, wherein the binding partner comprises an ExoSignal, wherein the ExoSignal is a sequence that associates with a LAMP2 protein.

[0404] Embodiment 11-55. The polynucleotide of embodiment 11-54, wherein the binding partner comprises at least 2, at least 3. at least 4, or at least 5 copies of an ExoSignal.

[0405] Embodiment 11-56. The polynucleotide of embodiment 11-54 or 11-55, wherein the ExoSignal comprises the amino acid sequence VKKDQAEPLEIRKFERQ (SEQ ID NO: 18).

[0406] Embodiment 11-57. The polynucleotide of any one of embodiments 11-51 to 11-56, wherein the EIEIDo comprises the amino acid sequence of any one of SEQ ID NOS: 18-20, or an amino acid sequence having at least 70% sequence identity thereto.

[0407] Embodiment 11-58. The polynucleotide of embodiment II-2, wherein the EIEIDo comprises a phosphatidylserine-binding polypeptide, or a domain or fragment thereof.

[0408] Embodiment 11-59. The polynucleotide of embodiment 11-58, wherein the phosphatidyl senne-binding polypeptide compnses a lactadherm polypeptide.

[0409] Embodiment 11-60. The polynucleotide of embodiment 11-59, wherein the lactadherin polypeptide comprises a ClC2 domain.

[0410] Embodiment 11-61. The polynucleotide of any one of embodiments 11-58 to 11-60, wherein the phosphatidylserine-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 70% sequence identity thereto.I l l

[0411] Embodiment 11-62. The polynucleotide of embodiment II-2, wherein the EIEIDo comprises a Platelet-Derived Growth Factor Receptor (PDGFR) polypeptide, or a domain or fragment thereof.

[0412] Embodiment 11-63. The polynucleotide of embodiment 11-62, wherein the PDGFR polypeptide, or domain or fragment thereof, comprises a human PDGFR polypeptide or a homologous PDGFR polypeptide from a non-human vertebrate species or an invertebrate species.

[0413] Embodiment 11-64. The polynucleotide of embodiment 11-63, wherein the PDGFR polypeptide comprises a human PDGFR polypeptide.

[0414] Embodiment 11-65. The polynucleotide of embodiment 11-63, wherein the PDGFR polypeptide comprises a PDGFR polypeptide from a non-human vertebrate species or an invertebrate species.

[0415] Embodiment 11-66. The polynucleotide of embodiment 11-63, wherein the homologous PDGFR polypeptide is selected from the group consisting of Spanish mole, brushtail possum, western clawed frog, Chinese alligator, and the common sole fish.

[0416] Embodiment 11-67. The polynucleotide of any one of embodiments 11-62 to 11-66, wherein the PDGFR polypeptide, or domain or fragment thereof, comprises a transmembrane domain of a PDGFR polypeptide.

[0417] Embodiment IT-68. The polynucleotide of any one of embodiments 11-62 to 11-67, wherein the PDGFR polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 22 and 228-232, or an amino acid sequence having at least 70% sequence identity thereto.

[0418] Embodiment 11-69. The polynucleotide of embodiment II-2, wherein the EIEIDo comprises a polypeptide capable of forming an amphipathic helix.

[0419] Embodiment 11-70. The polynucleotide of embodiment 11-69, wherein the EIEIDo is capable of inducing membrane fission by increasing membrane curvature when expressed in a cell.

[0420] Embodiment 11-71. The polynucleotide of embodiment 11-69 or 11-70. wherein the polypeptide capable of forming an amphipathic helix is isolated or derived from the group consisting of: M2 protein of influenza A, monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii, septum site-determining protein MinD from a bacterium (e.g., E. coli), peroxisomal membrane protein Peroxin 11 (Pexl Ip), phospholipid N-methyltransferase PmtA from Agrobacterium tumefaciens (AtPmtA), N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (MurG) from E. coli (EcMurG), EH domainbinding mitotic phosphoprotein (Epsin 1), amphiphysin from Drosophila, protein interacting with C kinase 1 (PICK1), ADP-ribosylation factor 1 (Arfl), diglucosyldiacylglycerol synthase (DGS) from Acholeplasma laidlawii, Endophilin Al, mammalian amphiphysin 2, Clathrin Assembly Lymphoid-Myeloid leukemia protein (CALM), or alpha-synuclein (a-synuclein).

[0421] Embodiment 11-72. The polynucleotide of any one of embodiments 11-69 to 11-71, wherein the polypeptide capable of forming an amphipathic helix comprises the amino acid sequence of any one of SEQ ID NOS: 23-40, or an amino acid sequence having at least 70% sequence identity’ thereto.

[0422] Embodiment 11-73. The polynucleotide of any one of embodiments 11-69 to 11-72, wherein the EIEIDo comprises at least two polypeptides capable of forming an amphipathic helix.

[0423] Embodiment 11-74. The polynucleotide of embodiment 11-73, wherein the at least two polypeptides are the same.

[0424] Embodiment 11-75. The polynucleotide of embodiment 11-73, wherein the at least two polypeptides are different.

[0425] Embodiment 11-76. The polynucleotide of any one of embodiments 11-73 to 11-75, wherein the at least two polypeptide capable of forming an amphipathic helix are isolated or derived from the group consisting of: M2 protein of influenza A, monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii, septum site-determining protein MinD from a bacterium (e.g., E. coli), peroxisomal membrane protein Peroxin 11 (Pexl lp), phospholipid N- methyltransferase PmtA from Agrobacterium tumefaciens (AtPmtA). N-acetylmuramyl- (pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (MurG) from E. coli (EcMurG), EH domain-binding mitotic phosphoprotein (Epsin 1), amphiphysin from Drosophila, protein interacting with C kinase 1 (PICK1), ADP-ribosylation factor 1 (Arfl), diglucosyldiacylglycerol synthase (DGS) from Acholeplasma laidlawii, Endophilin Al, mammalian amphiphysin 2, Clathrin Assembly Lymphoid-Myeloid leukemia protein (CALM), or alpha-synuclein (a-synuclein).

[0426] Embodiment 11-77. The polynucleotide of any one of embodiments 11-73 to 11-76, wherein the at least two polypeptides are linked in-frame.

[0427] Embodiment 11-78. The polynucleotide of embodiment 11-77, wherein the at least two polypeptides are linked by a linker.

[0428] Embodiment 11-79. The polynucleotide of embodiment 11-78, wherein the linker is a glycine serine linker.

[0429] Embodiment 11-80. The poly nucleotide of embodiment 11-78 or 11-79, wherein the linker comprises the sequence of any one of SEQ ID NOS: 209-217.

[0430] Embodiment 11-81. The polynucleotide of any one of embodiments 11-73 to 11-80, wherein the EIEIDo comprises the amino acid sequence of any one of SEQ ID NOS: 41-120, or an amino acid sequence having at least 70% sequence identity thereto.

[0431] Embodiment 11-82. The polynucleotide of any one of embodiments II- 1 to 11-81, wherein the fusion protein further comprises an endocytosis prevention motif (EPM).

[0432] Embodiment 11-83. The polynucleotide of any one of embodiments II- 1 to 11-82, wherein the antigenic polypeptide is derived from a viral protein.

[0433] Embodiment 11-84. The poly nucleotide of embodiment 11-83, wherein the viral protein is derived from 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), a coronavirus, influenza, a flavivirus, or orthopoxvirus.

[0434] Embodiment 11-85. The polynucleotide of embodiment 11-84, wherein the viral protein is derived from a coronavirus, wherein the coronavirus is Middle East respiratory syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), or SARS-CoV-2.

[0435] Embodiment 11-86. The polynucleotide of embodiment 11-85, wherein the protein is spike protein.

[0436] Embodiment 11-87. The polynucleotide of embodiment 11-84, wherein the viral protein is fusion (F) protein derived from hMPV, PIV3, or respiratory syncytial virus.

[0437] Embodiment 11-88. The poly nucleotide of embodiment 11-84, wherein the viral protein is:(a) glycoprotein E (gE) derived from N7N(b) glycoprotein H (gH), glycoprotein L (gL), or glycoprotein B (gB) derived from CMV; or(c) glycoprotein C (gC) or glycoprotein D (gD) derived from HSV1 or HSV2.

[0438] Embodiment 11-89. The polynucleotide of any one of embodiments II- 1 to 11-82, wherein the antigenic polypeptide is derived from a bacterial protein.

[0439] Embodiment 11-90. The polynucleotide of embodiment 11-89, wherein the bacterial protein is derived from an acne-causing bacterium, Staphylococcus, Borrelia, E. Coli, or Chlamydia.

[0440] Embodiment 11-91. The polynucleotide of embodiment 11-90, wherein the bacterial protein is:(a) DsAl derived from Cutibacterium acnes;(b) esxA or esxB derived from Staphylococcus;(c) OspA derived from Borrelia, optionally Borrelia burgdorferi;(d) FimH derived from E. Coli; or(e) major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), or OmcB derived from Chlamydia.

[0441] Embodiment 11-92. The poly nucleotide of any one of embodiments II- 1 to 11-82, wherein the antigenic polypeptide is derived from a parasite associated protein.

[0442] Embodiment 11-93. The polynucleotide of any one of embodiments II- 1 to 11-82, wherein the antigenic polypeptide is derived from a cancer associated protein.

[0443] Embodiment 11-94. The polynucleotide of embodiment 11-93, wherein the antigenic polypeptide is derived from a prostate cancer associated protein.

[0444] Embodiment 11-95. The polynucleotide of embodiment 11-94. wherein the prostate cancer associated protein is Six Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) or Prostate-specific membrane antigen (PSMA).

[0445] Embodiment 11-96. The polynucleotide of embodiment 11-93, wherein the antigenic polypeptide is derived from a melanoma associated protein.

[0446] Embodiment 11-97. The polynucleotide of embodiment 11-96, wherein the melanoma associated protein is Tyrosinase or transmembrane phosphatase with tensin homology protein (TPTE).

[0447] Embodiment 11-98. The polynucleotide of any one of embodiments II- 1 to 11-97, wherein the polynucleotide comprises RNA.

[0448] Embodiment 11-99. The polynucleotide of any one of embodiments II- 1 to 11-98, wherein the polynucleotide comprises mRNA.

[0449] Embodiment 11-100. The polynucleotide of any one of embodiments II-l to 11-99, wherein the polynucleotide comprises DNA.

[0450] Embodiment II- 101. The polynucleotide of any one of embodiments II- 1 to II- 100, wherein the polynucleotide, or the EIEIDo or fusion protein encoded by the polynucleotide: a) allow s for multivalent display of an antigenic polypeptide on an eVLP; b) allows for tailoring a lipid composition of eVLPs formed by cells expressing the fusion protein; c) enables altered modulation of eVLP surface presentation; d) enables incorporation of molecules, in addition to the antigenic polypeptide or fusion protein, into eVLPs formed by cells expressing the fusion protein; e) allows for controlling of the ratio of eVLP-associated and cell surface-associated antigens; and / or f) enables simultaneous production of a population of orthogonal eVLPs.

[0451] Embodiment 11-102. An EIEIDo comprising:(a) an envelope protein of a virus, or a domain or fragment thereof;(b) a tetraspanin polypeptide, or a domain or fragment thereof;(c) a binding partner of a tetraspanin polypeptide;(d) a Lysosome- Associated Membrane Protein 2 (LAMP2) polypeptide, or a domain or fragment thereof;(e) a binding partner of a LAMP2 polypeptide;(f) a phosphatidylserine-binding polypeptide, or a domain or fragment thereof;(g) a Platelet-Derived Grow th Factor Receptor (PDGFR) polypeptide, or a domain or fragment thereof; or(h) a polypeptide comprising an amphipathic helix; wherein (a) to (h) does not comprise a naturally occurring sequence.

[0452] Embodiment 11-103. A polypeptide encoded by the polynucleotide of any one of embodiments II- 1 to II- 101.

[0453] Embodiment 11-104. A fusion protein encoded by the polynucleotide of any one of embodiments II- 1 to II- 101.

[0454] Embodiment 11-105. A fusion protein, wherein the fusion protein has an amino acid sequence comprising any one of the sequences of SEQ ID NOS: 1-207 and 218-234, or a sequence having at least 70% sequence identity thereto.

[0455] Embodiment 11-106. A cell expressing on its surface the fusion protein, or a portion of the fusion protein, of embodiment 11-104 or 11-105.

[0456] Embodiment 11-107. A cell expressing on its surface the antigenic polypeptide of the fusion protein, or a portion of the antigenic polypeptide of the fusion protein, of embodiment II- 104 or 11-105.

[0457] Embodiment 11-108. An enveloped virus-like particle (eVLP) comprising a polypeptide encoded by the polynucleotide of any one of embodiments 11-1 to 11-101 or the fusion protein of embodiment 11-104 or 11-105.

[0458] Embodiment 11-109. An enveloped virus-like particle (eVLP) displaying on its surface the antigenic polypeptide, or a portion of the antigenic polypeptide, of the fusion protein of embodiment 11-104 or 11-105.

[0459] Embodiment II- 110. The eVLP of embodiment 11-108 or 11-109, wherein the diameter of the eVLP ranges from about 10 nm to about 1000 nm.

[0460] Embodiment II- 111. A vector comprising the polynucleotide of any one of embodiments II- 1 to II- 101.

[0461] Embodiment II- 112. The vector of embodiment II- 111, wherein the vector is a viral vector.

[0462] Embodiment II- 113. The vector of embodiment 11-111, wherein the vector is a non- viral vector.

[0463] Embodiment II- 114. The vector of embodiment II- 113, wherein the non- viral vector is a plasmid.

[0464] Embodiment II- 115. The vector of embodiment 11-113, wherein the non- viral vector comprises a lipid nanoparticle (LNP).

[0465] Embodiment II- 116. The vector of embodiment II- 113, wherein the non- viral vector comprises a lipid nanoparticle (LNP) and the polynucleotide comprises mRNA.

[0466] Embodiment II- 117. 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 the polynucleotide of any one of embodiments II- 1 to II- 101, the polypeptide of embodiment 11-102, the fusion protein of embodiment 11-104 or 11-105, the cell of embodiment 11-106 or 11-107, the eVLP of any one of embodiments 11-108 to II-l 10, or the vector of any one of embodiments Ill i 1 to 11-116.

[0467] Embodiment II- 118. A method of vaccinating a subject in need thereof, comprising administering to the subject an effective amount of the polynucleotide of any one of embodiments II- 1 to 11-101, the polypeptide of embodiment 11-102, the fusion protein ofembodiment 11-104 or 11-105, the cell of embodiment 11-106 or 11-107, the eVLP of any one of embodiments 11-108 to II- 110, or the vector of any one of embodiments II- 111 to II-l 16.

[0468] Embodiment II- 119. The method of embodiment II- 117 or 11-118, wherein the subject has been diagnosed with an infection or is at risk of being infected by a pathogen.

[0469] Embodiment 11-120. The method of embodiment II- 119, wherein the subject has been diagnosed with an infection with a virus, a bacterium, or a parasite, or is at risk of being infected by a virus, a bacterium, or a parasite.

[0470] Embodiment 11-121. The method of embodiment II- 117 or II- 118, wherein the subject has been diagnosed with cancer or is at risk of cancer.

[0471] Embodiment 11-122. The method of any one of embodiments II- 117 to 11-121, wherein the subject is a mammalian subject.

[0472] Embodiment 11-123. The method of embodiment 11-122, wherein the subject is a human subject.

[0473] Embodiment 11-124. The polynucleotide of any one of embodiments II- 1 to 11-101, the polypeptide of embodiment 11-102, the fusion protein of embodiment 11-104 or 11-105, the cell of embodiment 11-106 or 11-107, the eVLP of any one of embodiments 11-108 to II- 110, or the vector of any one of embodiments II- 111 to II-l 16 for use in the treatment or prevention of a disease or disorder.

[0474] Embodiment II-l 25. The polynucleotide of any one of embodiments II-l to II-l 01 , the polypeptide of embodiment 11-102, the fusion protein of embodiment 11-104 or 11-105, the cell of embodiment 11-106 or 11-107, the eVLP of any one of embodiments 11-108 to II-l 10, or the vector of any one of embodiments II-l 11 to II-l 16 for use in vaccination against a disease or disorder.

[0475] Embodiment 11-126. The polynucleotide of any one of embodiments II-l to II-l 01 , the polypeptide of embodiment 11-102, the fusion protein of embodiment 11-104 or 11-105, the cell of embodiment 11-106 or 11-107, the eVLP of any one of embodiments 11-108 to II- 110, or the vector of any one of embodiments II-l 11 to II-l 16 for use of embodiment 11-124 or 11-125, wherein the disease or disorder is an infection or a cancer.

[0476] Embodiment 11-127. The polynucleotide of any one of embodiments II-l to II-l 01 , the polypeptide of embodiment 11-102, the fusion protein of embodiment 11-104 or 11-105, the cell of embodiment 11-106 or 11-107, the eVLP of any one of embodiments 11-108 to II-l 10. or thevector of any one of embodiments II- 111 to II- 1 16 for use of embodiment 11-124 or 11-125, wherein the infection is a viral infection, a bacterial infection, or a parasitic infection.

[0477] Embodiment 11-128. The polynucleotide of any one of embodiments II-l to 11-101, the polypeptide of embodiment 11-102, the fusion protein of embodiment 11-104 or 11-105, the cell of embodiment 11-106 or 11-107, the eVLP of any one of embodiments 11-108 to 11-110. or the vector of any one of embodiments II- 111 to II- 116 for use of embodiment 11-124 or 11-125, wherein the cancer is prostate cancer or melanoma.

[0478] Embodiment 11-129. Use of the polynucleotide of any one of embodiments II-l to II- 101, the polypeptide of embodiment 11-102, the fusion protein of embodiment 11-104 or 11-105, the cell of embodiment 11-106 or 11-107, the eVLP of any one of embodiments 11-108 to II-l 10, or the vector of any one of embodiments II-l 11 to II-l 16 for manufacture of a medicament for the prevention or treatment of a disease or disorder.

[0479] Embodiment 11-130. Use of the polynucleotide of any one of embodiments II-l to II- 101, the polypeptide of embodiment 11-102, the fusion protein of embodiment 11-104 or 11-105, the cell of embodiment 11-106 or 11-107, the eVLP of any one of embodiments 11-108 to II-l 10, or the vector of any one of embodiments II-l 11 to II-l 16 for manufacture of a medicament for vaccination against a disease or disorder.

[0480] Embodiment 11-131. The use of embodiments 11-129 or 11-130, wherein the disease or disorder is an infection or a cancer.

[0481] Embodiment 11-132. The use of embodiment 11-131, wherein the infection is a viral infection, a bacterial infection, or a parasitic infection.

[0482] Embodiment 11-133. The use of embodiment 11-131, wherein the cancer is prostate cancer or melanoma.

[0483] Embodiment 11-134. An ESCRT-independent eVLP inducing domain (EIEIDo) comprising: a. a tetraspanin polypeptide, wherein the tetraspanin polypeptide is a chimeric tetraspanin polypeptide; b. a tetraspanin polypeptide derived from CD63, wherein the tetraspanin polypeptide comprises a modification in an endosome- targeting signal in CD63; or c. at least two polypeptides capable of forming an amphipathic helix.

[0484] Embodiment 11-135. The EIEIDo of embodiment 11-134, comprising a tetraspanin polypeptide, wherein the tetraspanin polypeptide is a chimeric tetraspanin polypeptide, andwherein the chimeric tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 184-207, or an amino acid sequence having at least 70% sequence identity thereto.

[0485] Embodiment 11-136. The EIEIDo of embodiment 11-134, comprising a tetraspanin polypeptide derived from CD63. wherein the tetraspanin polypeptide comprises a modification in an endosome-targeting signal in CD63.

[0486] Embodiment 11-137. The EIEIDo of embodiment 11-136, comprising a modification of a tyrosine (Y) in an endosome-targeting signal in CD63.

[0487] Embodiment 11-138. The EIEIDo of embodiment 11-137, comprising an amino acid substitution of a tyrosine (Y) to alanine (A) in an endosome-targeting signal in CD63.

[0488] Embodiment 11-139. The EIEIDo of any one of embodiments 11-136 to 11-138, wherein the modification is at a position corresponding to position 235 relative to SEQ ID NO: 11.

[0489] Embodiment 11-140. The EIEIDo of embodiment 11-135, comprising at least two polypeptides capable of forming an amphipathic helix, wherein the at least two polypeptide capable of forming an amphipathic helix are isolated or derived from the group consisting of: M2 protein of influenza A, monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii, septum site-determining protein MinD from a bacterium (e.g., E. coli), peroxisomal membrane protein Peroxin 11 (Pexl Ip), phospholipid N-methyltransferase PmtA from Agrobacterium tumefaciens (AtPmtA), N-acetylmuramyl-(pentapeptide) pyrophosphoryl- undecaprenol N-acetylglucosamine transferase (MurG) from E. coli (EcMurG), EH domainbinding mitotic phosphoprotein (Epsin 1), amphiphysin from Drosophila, protein interacting with C kinase 1 (PICK1), ADP-ribosylation factor 1 (Arfl), diglucosyldiacylglycerol synthase (DGS) from Acholeplasma laidlawii, Endophilin Al, mammalian amphiphysin 2, Clathrin Assembly Lymphoid-Myeloid leukemia protein (CALM), or alpha-synuclein (a-synuclein).

[0490] Embodiment 11-141. The EIEIDo of embodiment 11-140, wherein the at least two polypeptides are linked in-frame.

[0491] Embodiment 11-142. The EIEIDo of embodiment 11-141, wherein the at least two polypeptides are linked by a linker.

[0492] Embodiment 11-143. The EIEIDo of embodiment 11-142, wherein the linker is a glycine serine linker.

[0493] Embodiment 11-144. The EIEIDo of any one of embodiments 11-140 to 11-143, wherein the EIEIDo comprises the amino acid sequence of any one of SEQ ID NOS: 41-120, or an amino acid sequence having at least 70% sequence identity' thereto.EXAMPLESExample 1A: Generation and Characterization of MERS Enveloped Virus-Like Particles (eVLPs) Using ESCRT-independent eVLP Inducing Domains

[0494] To develop ESCRT-independent eVLP inducing domains that facilitate production of eVLPs. MERS-1227 (SEQ ID NO: 5). was utilized as a model antigen to evaluate potential ESCRT-independent eVLP Inducing Domains for vaccine development. MERS-1227, a truncated MERS spike protein derived from the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV), has been extensively characterized for immunogenicity in various animal models (see, for example, Powell el al. bioRxiv 2024.07.01.601243). This antigen, truncated at position 1227, in a variety of forms, including as an mRNA-delivered antigen with a transmembrane (TM) domain and C-terminal domain (CTD) is immunogenic in mouse models. Accordingly, MERS-1227 with a TM domain served as a representative model antigen for initial evaluation.

[0495] To accommodate the diverse requirements of different ESCRT-independent eVLP inducing domain classes, we designed 35 initial constructs using four backbone variations from the MERS spike protein and six insertion sites. This was designed to enable efficient production of ESCRT-independent eVLP inducing domain proteins utilizing the MERS-1227 scaffold while retaining the required protein features. FIGS. 8A-8D depict linear schematics illustrating the four backbone variations including the different insertion sites. SEQ ID NOS: 1-4 comprise the amino acid sequences of the four backbone variations. Each of the 35 initial constructs were included within one of the four backbone variations at one of the different insertion sites.

[0496] The sequences of the 35 initial ESCRT-independent eVLP inducing domains along with each construct's backbone usage, insertion site, insertion sequence and plate layout are detailed in Table 9 and depicted in FIGS 8A-8D.Table 9: Plate layout and construct information

[0497] The endocytosis prevention motif (EPM) derived from the murine Fc gamma receptor FcgRII-Bl cytoplasmic tail was incorporated into all constructs except the C1C2 domain construct (#16 in Table 9). The absence of a transmembrane domain in the C1C2 domain construct precluded any portion of it from residing within eVLPs, thus excluding a site for EPM.

[0498] Individual ESCRT-independent eVLP inducing domain sequences were fused to the target site in the MERS Spike protein ectodomain using a plasmid (pTwist CMV BG WPRE Neo-Nstre-MERS1227-EPM-EABR), initially linearized via PCR using primers Nstrep-MERS S ecto FWD (TGA TCT AGA AGT TGT CTC CTC CTG CAC TGA CT; SEQ ID NO: 121) and REV (GGG ACT ACC CCC TGT GGA GCC; SEQ ID NO: 122). ESCRT-independent eVLP inducing domain amino acid sequences (Table 9) were codon optimized using the IDT codon optimization tool and synthesized by IDT. The Gibson assembly cloning strategy was employed,whereby 15-base pair overlaps with the 3’ and 5’ ends of the linearized vector DNA were added to the 5’ and 3’ ends of the ESCRT-independent eVLP-inducing domain (EIEIDo) gene fragments, respectively. Subsequently, 10 ng of the EIEIDo genes were incubated with 20 ng of linearized vector and 0.5 pl of In-Fusion® Snap Assembly Master Mix (Takara) at 50°C for 15 minutes. Subcloned ESCRT-independent eVLP inducing domain constructs were amplified using the GeneJET Plasmid Miniprep Kit (Thermo Scientific), and the sequence of each plasmid was confirmed via NanoPore sequencing at Plasmidsaurus.

[0499] MERS Spike-ESCRT-independent eVLP inducing domain Cell Transfection, Dot Blot, and Cell ELISA: HEK293T cells were seeded at 30,000 cells / well on a 96-well tissue culture plate (Greiner Bio-One). After approximately 6 hours, 100 ng of EIEIDo plasmid DNA was mixed with 5 pl of serum-free DMEM medium (Thermo Scientific) and 0.15 pl of BioT transfection reagent (Morganville Scientific), follow ed by a 5-minute incubation at room temperature. The transfection cocktail was then added to each well containing seeded HEK293T cells. Approximately 18 hours post-transfection, the cell medium was replaced with fresh DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin. After 72 hours post-transfection, cell culture supernatant was collected and centrifuged at lOOOxg for 10 minutes to remove live cells and cell debris. Subsequently, 1.5 pl of supernatant was blotted on a nitrocellulose membrane (Bio-Rad) and allowed to dry completely for 30 minutes. The membrane was then incubated with blocking solution (5% non-fat milk in PBST) for an hour, followed by incubation with anti-MERS spike antibody D12 at 1 :1000 dilution (in blocking solution) for another hour. After w ashing with PBST three times (5 minutes each time), the membrane was incubated with Goat Anti-Human IgG Fc HRP-conjugated secondary antibody at 1 :4000 dilution (in blocking solution) for an additional hour. Following three more PBST w ashes, the membrane w as incubated with Pierce™ ECL Western Blotting Substrate Mix (3ml luminol / enhancer plus stable peroxide buffer, Thermo Scientific) for 3 minutes. Dot blot images were captured using an iBright™ CL750 Imaging System (Thermo Scientific), and the intensity of each dot w as quantified using ImageJ image analyzer (National Institutes of Health).

[0500] For high-throughput screening, ESCRT-independent eVLP inducing domain constructs were delivered to HEK293T cells as plasmid DNA. After 72 hours post-transfection, the eVLP levels generated from each construct in the supernatant were quantified using dot blot analysis (FIG. 9). and expression levels were measured by cell ELISA (FIG. 10). MERS spike antibody D12 served as the detection antibody in both assays. As positive controls and ESCRT-dependentcomparators, human CEP55 ESCRT and ALIX-binding regions (EAB Rs) and truncated EABRs, ESCRT-dependent proteins, were used (Construct #36 and Construct #37, Table 9). To establish the baseline level of MERS protein secretion, a MERS spike construct lacking the EABR region (Construct #38, MERS FL EABR-null control in FIG. 9) was employed.

[0501] Among the different classes of ESCRT-independent eVLP inducing domain constructs, constructs comprising the C1C2 domain and tetraspanins including CD9 and CD81 (Constructs #16, 29, and 30 in FIG. 9) exhibited superior performance compared to the positive controls EABR and truncated EABR (Constructs #36 and 37 in FIG. 9). Notably, while the structure of the C1C2 domain lacks a transmembrane domain, it cannot be definitively concluded whether some MERS-C1C2 protein was secreted rather than tethered to eVLPs. Constructs comprising viral Envelope proteins and the HCMV cytoplasmic tail did not induce detectable levels of eVLPs. While direct fusion to Lamp2a or 2b did not appear to sort MERS spike protein to eVLPs. the Lamp2a-binding motif ExoSignal (Construct #17 in FIG. 9) elicited a moderate level of eVLP. However, the co-presentation of ExoSignal with EABR significantly reduced eVLP levels, suggesting potential interference between ESCRT-dependent and ESCRT-independent domains within the same construct.

[0502] Constructs comprising the PDGFR transmembrane domain and / or several amphipathic helices exhibited moderate levels of eVLP generation. Notably, the influenza A M2 amphipathic helix failed to elicit eVLP formation when the native M2 transmembrane domain was replaced with that of the MERS spike protein or when the cytoplasmic tail outside of the amphipathic helix was truncated.

[0503] Cell ELISA was conducted concurrently with dot blot analysis. After collecting cell culture supernatant, HEK293T cells were fixed with fixative (80% acetone with 20% PBS) for 10 minutes. Following fixative removal, the plate was left in a fume hood for 30 minutes to allow complete evaporation of residual fixative. Subsequently, the plate was blocked with 300 pl of 3% non-fat milk (in PBST) for an hour. After three washes with 300 pl PBST. the plate was incubated with D12 antibody at 1: 1000 dilution in antibody diluent (0.05% Tween-20 and 0.1% bovine serum albumin in DPBS) for an hour. The plate was then washed six times and incubated with Goat Anti-Human IgG Fc HRP-conjugated secondary antibody at 1:4000 dilution (in antibody diluent) for another hour. Following another six washes to remove the secondary antibody completely, 50 pl of 1 -step Turbo TMB solution (Thermo Scientific) was added to each w ell. After 5 minutes of incubation, 50 pl of stop solution (Thermo Scientific) was added toterminate the reaction, and absorbance at 450 nm was measured using a BioTek Synergy Hl Multimode Reader (Agilent).

[0504] Cell ELISA data (FIG. 10) revealed significant variability in expression levels among ESCRT-independent eVLP inducing domains, indicating their significant impact on intracellular expression levels. The positive correlation observed between dot blot intensity and cell ELISA signal levels underscores the importance of protein expression in driving eVLP formation.

[0505] To further delve into the eVLPs generated from diverse classes of ESCRT-independent eVLP inducing domains, a selection of 7 constructs was chosen for isolating eVLPs from transfected Expi293F cell culture using the Strep-Tactin purification as depicted in FIG. 11. The 7 constructs include MERS S ecto-M2 TM-CT, MERS ecto-ClC2, MERS_FL-EPM-ExoSignal, MERS FL-EcMurG AH-EPM, MERS FL-Drosophila amphiphysin_AH-EPM, MERS FL-EPM CD81, and MERS ecto-PDGFR-EPM.

[0506] The intensity of bands observed in purified eVLPs (FIGS. 12A-12B) mirrored that of the dot blot (FIG. 9), implying that the dot intensity in dot blots accurately reflects the quantity of eVLPs generated by the ESCRT-independent eVLP inducing domains. The fusion proteins ran at the anticipated molecular weight, as seen in FIGS. 12A-12B. As seen in the gel (FIG. 12B), the purified eVLPs contain predominantly the MERS spike protein of interest with minimal host cell protein contamination. Approximate similar rank order of the ability of various EIEIDos to facilitate eVLP formation was determined from this analysis and was consistent with data presented in FIG. 9 and FIG. 10.

[0507] The size of purified eVLPs was assessed via dynamic light scattering (DLS). As seen in FIG. 13, eVLPs generated from the ESCRT-independent eVLP inducing domain ranged from approx, radiuses of 40 to 80nm (FIG. 13). eVLPs were purified via an N-terminal Strep Tag 2 using StrepTactin® resin. ERD-based purified eVLPs show a range of sizes consistent with eVLPs, suggesting the purified proteins are embedded in, or associated with, a lipid bilayer and entire eVLPs are being co-purified. Transmission electron microscopy (TEM) images, including representative frames, are provided in FIGS. 14A-14F for punfied eVLPs induced by Lactadherin C1C2 domain (FIG. 14A), ExoSignal (FIG. 14B), EcMurG amphipathic helix (FIG. 14C), Drosophila amphiphysin amphipathic helix (FIG. 14D), CD81 (FIG. 14E), or human PDGFR transmembrane domain (FIG. 14F) fused to MERS spike protein. Collectively , these data demonstrate that fusion proteins comprising the listed ESCRT-independent eVLP inducing domain 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.Example IB: Non-human orthologs of PDGFR transmembrane domain, CD81, and CD9 induce eVLP formation

[0508] To test whether PDGFR transmembrane domain (TM), CD81. and CD9 derived from non-human species induce eVLP formation, non-human PDGFR transmembrane domains, CD81s, and CD9s with various degrees of homology to respective human sequences were fused to the C terminus of MERS spike protein. CD81 sequences derived from Atlantic canary, Florida worm lizard, Adelie penguin, Common toad, and Orbiculate cardinalfish (SEQ ID NOS: 218- 222, respectively) were tested. CD9 sequences derived from Aardvark, Diamondback terrapin, MacQueen's bustard, Green anole, and Common wombat (SEQ ID NOS: 223-227, respectively) were tested. PDGFR transmembrane domains derived from PDGFR protein of Spanish mole, brushtail possum, western clawed frog, Chinese alligator, and common sole fish (SEQ ID NOS: 228-232. respectively) were tested. Human HEK293T cells were transfected with constructs containing PDGFR transmembrane domain, CD81, and CD9 from non-human species fused to MERS spike protein. The eVLP level in corresponding cell culture supernatant was measured by dot blot using D12 antibody as the detection antibody.

[0509] As shown in FIG. 16A, non-human CD81 sequences demonstrated significant eVLP inducing activity, although the degree of activity was lower compared to that induced by human CD81.

[0510] As shown in FIG. 16B, significant eVLP inducing activity was observed for Green anole, Common wombat, and Diamondback terrapin CD9, with Green anole CD9 showing a degree of eVLP formation comparable to human CD9. CD9 from MacQueen’s bustard and Aardvark showed little to no eVLP inducing activity.

[0511] As shown in FIG. 16C, human PDGFR TM domain demonstrated significant eVLP inducing activity. Surprisingly, non-human PDGFR TM domains, in particular TM domain derived from common sole fish PDGFR, demonstrated higher degrees of eVLP inducing activity than human PDGFR TM domain.Example 2: EIEIDo drives secretion of membrane-bound WNV NS1 homodimer

[0512] Different classes of EIEIDos may use varied mechanisms of action to drive eVLP formation; therefore, several different WNV NS1 backbones were designed to accommodateeach class of EIEIDos, as depicted in FIGS. 17D-17G. Each construct's backbone usage, EIEIDo insertion site, and insertion sequence are detailed in Table 10 below.Table 10: Details of exemplary constructs in FIG. 18

[0513] HEK293T cells were transfected with lOOng of each construct, and the presence of membrane-bound WNV NS1 protein in the cell supernatant was detected by a WNV NS 1- specific antibody 22NS1 at Ipg / ml. As shown in FIG. 18, WNV NS1 homodimer (WNV dNSl) was better expressed across different EIEIDos compared to WNV NS1 monomer (WNV mNSl). However, the cell-anchored control also displayed a moderate level of signal, suggesting that thetransmembrane domain from measles virus hemagglutinin used to anchor the WNV NS1 protein to the plasma membrane may drive spontaneous release of NS1 protein into the supernatant through unknown mechanisms. Therefore, to reduce spontaneous release of NS1 protein, additional transmembrane domains were tested, including the transmembrane domains from Epstein-Barr Virus (EBV) gp220 protein, Sendai Virus F protein. SARS-CoV-2 E protein, and Human Cytomegalovirus Virus gH protein. Constructs containing these transmembrane domains are detailed in Table 11 below.Table 11: Details of exemplary constructs in FIG. 19

[0514] In a separate comparative study with the human ortholog, transmembrane domain of common sole fish PDGFR showed a greater tendency to drive eVLP formation. Therefore, this PDGFR transmembrane domain was also used to replace the human sequence in this experiment (WNV dNSl-PDGFR_2, Table 11 and FIG. 19). As in FIG. 18, where MinD.DGS tandem amphipathic helix shows a signal lower than the cell-anchored control, additional screening withdifferent TM domain (see the list of the TM domain in Table 10) was performed. It is shown that among different TM domains, WNV dNSl-CA2 (Epstein-Barr Virus gp220) seemed to better curb the signal of the cell-anchored control (FIG. 19). Similar to a previous study, the TM domain of common sole fish PDGFR also enhanced the signal from this class of EIEIDo with WNB NS1 homodimer. Interestingly, TM domain did not show the same effect with MinD.DGS tandem amphipathic helix when tested with 4 different TM domains.Example 3: Membrane-bound WNV-JEV NS1 heterodimer was released from two different cell types

[0515] Schematic of different tetraspanin-based vaccine designs intended to improve B and T cell responses against engineered antigens are provided in FIG. 20. Tetraspanin proteins naturally make rafts on the plasma membrane and enable eVLP production through physical bending of the membrane. To commandeer this activity without direct fusion, mutations in the antigen TM domain or cytoplasmic tail may be made to facilitate interactions with tetraspanin proteins (FIG. 20, top). Additionally, the extracellular domain of tetraspanin proteins can be swapped, producing chimeric tetraspanin proteins. These chimeras may be designed to have differing activities including improved co-stimulation of T cells, etc. (FIG. 20, left). The C- terminal portion of tetraspanin proteins may be mutated to interact directly with MHC molecules. For example, a PDZ domain, could be added C-terminally of the tetraspanin to promote MHC association (FIG. 20, bottom left). Increased MHC presentation through improved incorporation of MHC into tetraspanin enriched eVLPs could facilitate improved T cell co-stimulation. To encourage MHC incorporation into tetraspanin enriched eVLPs, tetraspanin protein transmembrane domains may be engineered to promote interaction with the MHC molecules (FIG. 20, right). Improved MHC incorporation into eVLPs (through tetraspanin engineering) may promote improved MHC presentation of intracellular antigens (not directly fused to tetraspanin proteins) and improve co-stimulation of T cell responses.

[0516] A total of 70 tetraspanin constructs, as described in Table 12, were screened in HEK293T cells. 30,000 HEK293T cells were seeded and transfected with tetraspanins. The tetraspanin was directly fused to WNV-JEV NS1 (an NS1 heterodimer composed of NS1 protein from West Nile Virus (WNV) and Japanese Encephalitis Virus (JEV)) or co-transfected with WNV-JEV NS1 equipped with an EWI-F transmembrane domain. As depicted in FIG. 21A andFIG. 21B, respectively, following signal peptide (Sip Pep) and StrepTag II (Strep) sequence. West Nile Virus NS1 protein is fused to Japanese Encephalitis Virus NS1 by a SGSG linker. To anchor this NS1 heterodimer to membrane surface, the transmembrane domain (TM) of Epstein- Barr Virus gp220 protein was added to the C terminus of JEV NS1 protein. Tetraspanin was then added to the C terminus of the TM domain to drive eVLP formation (FIG. 21A). while for some designs, the EPM sequence was added betw een TM domain and tetraspanin (FIG. 21B). The backbone and insertion site used by each tetraspanin design is shown in Table 12 and 13.

[0517] Details of the constructs and various controls that were tested are provided in Table 12 below.Table 12: Details of exemplary constructs in FIGS. 22A and 22B

[0518] In Table 12, which corresponds to the dot plots presented in FIGS. 22A and 22B, well numbers 1 through 5 contained control constructs in absence of tetraspanin. Well numbers 6 through 79 contained constructs comprising human, non-human tetraspanins, and tetraspanin chimeras directly fused to WNV-JEV NS1 protein. An EPM was also added to constructs in well numbers 55, 56, and 57, but not in other direct fusion constructs. Well numbers 80 through 105 contained constructs comprising human tetraspanins and their chimeras without NS1 direct fusion. Constructs comprising NS1 with the EWI-F transmembrane domain and cytoplasmic tail (NS1_EWI-F_TM_CPT no EDN) fused to its C terminus were added in wells 106 through 131 , whereas constructs comprising NS1 with only the EWI-F transmembrane domain was added to wells 132 through 157 (NS1_EWI-F_TM no EDN). Wells 160 and 161 are constructs comprising NS1 protein directly fused to human CD9 and CD81, respectively, with a PDZ1 domain; whereas wells 158 and 159 are constructs comprising human CD9 and CD81, respectively, with a PDZ1 domain without NS1 direct fusion.

[0519] The presence of NS1 was detected by a flavivirus NS1 cross-reactive antibody 1G5.3 (FIG. 22A) and a WNV NS 1 -specific antibody 22NS1 (FIG. 22B). Due to the discrepancy between the results given by these antibodies, all positive hits, along with controls, in the screening were selected and screened again in Expi293F cells. Details of constructs that were screened in Expi293F cells are provided in Table 13 below.Table 13: Details of exemplary constructs in FIGS. 23A-C and FIG. 24

[0520] 22NS1 antibody was again used to detect the presence of WNV NS1 protein in the cell supernatant (FIG. 23A), and JA12 antibody was used to detect JEV NS1 protein in the WNV- JEV NS1 heterodimer (FIG. 23B). As expected, the results from 22NS1 and JA12 antibodies mirror each other, confirming the presence of the NS 1 heterodimer. To further confirm the dot blot results, a sandwich ELISA was performed. In the sandwich ELISA, membrane-bound NS1 protein was captured by 22NS1 antibody in human Fc and then detected by the same antibody but in mouse Fc. As shown in FIG. 23C, the ELISA results reflect that of the dot blot using 22NS1 antibody. To detect if human HLA class I was co-packaged in tetraspanin-driven eVLPs. a separate dot blot was performed using the same cell supernatant from Expi293F cells. A mouse anti-human HLA-ABC antibody was used to recognize human HLA-I. As shown in FIG. 24, despite a high background noise, some of the dots show an intensity higher than the mock transfection control (FIG. 24; dot no 32), showing some HLA is present in the tetraspanin-driven eVLPs.Example 4: Immunogenicity of EIEIDo-delivered MERS spike protein

[0521] To analyze the impact of EIEIDos on antibody response in vivo, mice (3 animals in each group) were immunized with MERS spike protein fused to different EIEIDos including TM domain of human PDGFR, C1C2 domain. EcMurG amphipathic helix, CD81, and ExoSignal. Controls like cell-anchored, human EABR, and blank were also included. Mice were immunized on day 0 and day 21, and serum antibody response was analyzed on day 21 and day 35 to test the immunogenicity after the prime (day 0) and boost (day 21) shots. The antibody response was analyzed at 1:250 dilution by ELISA. As shown in FIG. 25, cell-anchored control and EABR showed the highest titer after immunization, in both day 21 and day 35 sera. Based on a previous immunogenicity study with MERS spike protein, the response was likely saturated at this dilution. Compared among classes of EIEIDos, PDGFR TM domain induced the highest response in vivo, as measured in day 35 sera, even though the C1C2 and CD81 showed a muchhigher signal in dot blots experiments, suggesting that in vivo antibody response data and dosing are important factors to consider in assessing immunogenicity.

Claims

CLAIMS1. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises:(a) an antigenic polypeptide; and(b) a polypeptide comprising an ESCRT-independent eVLP inducing domain (EIEIDo).

2. The polynucleotide of claim 1, wherein the EIEIDo comprises:(a) an envelope protein of a virus, or a domain or fragment thereof;(b) a tetraspanin polypeptide, or a domain or fragment thereof;(c) a binding partner of a tetraspanin polypeptide;(d) a Lysosome- Associated Membrane Protein 2 (LAMP2) polypeptide, or a domain or fragment thereof;(e) a binding partner of a LAMP2 polypeptide;(f) a phosphatidylserine-binding polypeptide, or a domain or fragment thereof;(g) a Platelet-Derived Growth Factor Receptor (PDGFR) polypeptide, or a domain or fragment thereof; or(h) a polypeptide comprising an amphipathic helix.

3. The polynucleotide of claim 2, wherein the EIEIDo comprises an envelope protein of a virus, or a fragment thereof.

4. The polynucleotide of claim 3, wherein the EIEIDo comprises the cytoplasmic tail of the envelope protein.

5. The polynucleotide of any one of claims 2-4, wherein the virus is a coronavirus.

6. The polynucleotide of any one of claims 2-5, wherein the coronavirus is a Gammacoronavirus, a Betacoronavirus, or an Alphacoronavirus.

7. The polynucleotide of any one of claims 2-6, wherein the virus is selected from a group consisting of: Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, avian infectious bronchitis virus (AIBV), mouse hepatitis virus (MHV), and transmissible gastroenteritis virus (TGEV).

8. The polynucleotide of any claim of claims 2-7, wherein the envelope protein comprises the amino acid sequence of any one of SEQ ID NOS: 6-9, or an amino acid sequence having at least 70% sequence identity7thereto.

9. The polynucleotide of claim 2, wherein the EIEIDo comprises a tetraspanin polypeptide, or a domain or fragment thereof.

10. The polynucleotide of claim 9. wherein the tetraspanin polypeptide comprises a human tetraspanin polypeptide or a homologous tetraspanin polypeptide from a non-human vertebrate species or an invertebrate species.

11. The polynucleotide of claim 10, wherein the tetraspanin polypeptide comprises a human tetraspanin polypeptide.

12. The polynucleotide of claim 10, wherein the tetraspanin polypeptide comprises a tetraspanin polypeptide from a non-human vertebrate species or an invertebrate species.

13. The polynucleotide of claim 12, wherein the non-human vertebrate species or invertebrate species is selected from the group consisting of: Spanish mole, brushtail possum, western clawed frog. Chinese alligator, common sole fish, Atlantic canary, Florida worm lizard, Adelie penguin, Common toad, Orbiculate cardinalfish. Aardvark, Diamondback terrapin, MacQueen's bustard, Green anole, Common wombat, Budorcas taxicolor, Patagioenas fasciata, Maylandia zebra. Hypanus sabinus, Echinops telfairi, Pan troglodytes, Lagopus leucura, Thamnophis elegans, Ornithorhynchus anatinus, Etheostoma spectabile, Carassius Carassius, Pleuronectes platessa, Monodon monoceros, Motacilla alba alba, Syngnathus typhle, Xiphophorus couchianus, Lonchura striata, Meriones unguiculatus. Vicugna pacos, Podarcis murcdis, Sinocyclocheilus anshuiensis, Sorex fumeus, Ursus arctos, Myotis davidii, Bos taunts, Gopherus flavomarginatus, Orycteropus afer of er, Phasianus colchicum, Actinia equina, Orbicella faveolata, Daphnia carinata, Microtus oregoni, Petaurus breviceps papuanus. Gallus gallus. Myotis lucifugus. Malaclemys terrapin pileata, Bufo bufo , Zonotrichia leucophrys gambelii, Anolis sagrei, Leopardus geoffroyi, Manacus candei, and Sapajus apella.

14. The polynucleotide of any one of claims 9-13, wherein the tetraspanin polypeptide is derived from a protein selected from the group consisting of: CD9, CD63, CD81, CD82. CD37,CD53, CD151, CD231, tetraspanin (TSP)-l, TSP-2, TSP-3, TSP-4, TSP-5, TSP-6, TSP-9, TSP- 11, and proteins encoded by genes TSPAN1-TSPAN33.

15. The polynucleotide of claim 14, wherein the tetraspanin polypeptide is derived from CD9.

16. The polynucleotide of claim 15, wherein the tetraspanin polypeptide is derived from human CD9 or CD9 from Aardvark. Diamondback terrapin, MacQueen's bustard, Green anole, Common wombat. Budorcas taxicolor, Patagioenas fasciata, Maylandia zebra, Hypanus sabinus, Echinops telfairi, Pan troglodytes, Lagopus leucura, Thamnophis elegans, Ornithorhynchus anatinus, Etheostoma spectabile, Carassius Carassius, Pleuronectes platessa, Monodon monoceros, Molacilla alba alba, or Syngnathus typhle.

17. The polynucleotide of claim 15, wherein the tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 10, 138-152, and 223-227, or an amino acid sequence having at least 70% sequence identity thereto.

18. The polynucleotide of claim 14, wherein the tetraspanin polypeptide is derived from CD63.

19. The polynucleotide of claim 18, wherein the tetraspanin polypeptide is derived from human CD63 or CD63 from Actinia equina, Orbicella faveolata, Daphnia carinata, Microtus oregoni. Petaurus breviceps papuanus, Gallus gallus, Myotis lucifugus, Malaclemys terrapin pileata, Bufo bufo, Zonotrichia leucophrys gambelii, Sorex fumeus , Anolis sagrei. Leopardus geoffroyi, Manacus candei, or Sapajus apella.

20. The polynucleotide of claim 18 or 19, wherein the tetraspanin polypeptide comprises a modification in an endosome-targeting signal in CD63.

21. The polynucleotide of claim 20, wherein the endosome-targeting signal comprises a modification of a tyrosine (Y) in the endosome-targeting signal in CD63, wherein the modification is an amino acid substitution.

22. The polynucleotide of claim 20 or 21, comprising a modification of a glutamate (E) in the endosome-targeting signal in CD63.

23. The polynucleotide of any one of claims 20-22, comprising a modification of a valine (V) in the endosome-targeting signal in CD63.

24. The polynucleotide of any one of claims 20-23, comprising a modification of a methionine (M) in the endosome-targeting signal in CD63.

25. The polynucleotide of any one of claims 20-24, comprising an amino acid substitution of tyrosine (Y) to alanine (A) in the endosome-targeting signal in CD63.

26. The polynucleotide of any one of claims 18-20, comprising a modification at a position corresponding to position 235, 236, 237, and / or 238 relative to SEQ ID NO: 11.

27. The polynucleotide of claim 26, comprising a modification at a position corresponding to position 235 relative to SEQ ID NO: 11.

28. The polynucleotide of claim 27, wherein the modification is an amino acid substitution of tyrosine (Y) to alanine (A).

29. The polynucleotide of any one of claims 18-28, wherein the tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 11 and 168-183. or an amino acid sequence having at least 70% sequence identity thereto.

30. The polynucleotide of claim 14, wherein the tetraspanin polypeptide is derived from CD81.

31. The polynucleotide of claim 30, wherein the tetraspanin polypeptide is derived from human CD81 or CD81 from Atlantic canary , Florida worm lizard, Adelie penguin, Common toad, Orbiculate cardinalfish, Xiphophorus couchianus, Lonchura striata. Meriones unguiculatus. Vicugna pacos, Hypanus sabinus. Podarcis muralis, Sinocyclocheilus anshuiensis. Sorex fumeus. Ursus arctos, Myotis davidii, Patagioenas fasciata. Bos taurus. Gopherus flavomarginatus, Orycteropus afer afer, or Phasianus colchicus.

32. The polynucleotide of claim 30 or 31, wherein the tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 12, 153-167, and 218-222, or an amino acid sequence having at least 70% sequence identity thereto.

33. The polynucleotide of any one of claims 9-32, wherein the EIEIDo comprises a chimeric tetraspanin polypeptide, wherein the chimeric tetraspanin polypeptide comprises a first tetraspanin polypeptide, wherein a domain in that first tetraspanin polypeptide is substituted with a domain from a second tetraspanin polypeptide.

34. The polynucleotide of claim 33, wherein the first and second tetraspanin polypeptides are different.

35. The polynucleotide of claim 33 or 34, wherein the first and second tetraspanin polypeptides are independently selected from the group consisting of: CD9, CD63, CD81, CD82, CD37, CD53, CD151, CD231, tetraspanin (TSP)-l, TSP-2, TSP-3, TSP-4, TSP-5, TSP-6, TSP-9, TSP-11, proteins encoded by genes TSPAN1-TSPAN33, and domains or fragments thereof.

36. The polynucleotide of any one of claims 33-35, wherein the chimeric tetraspanin polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 184-205, or an amino acid sequence having at least 70% sequence identity thereto.

37. The polynucleotide of any one of claims 9-35, wherein the EIEIDo further comprises a PDZ1 domain.

38. The polynucleotide of claim 37, wherein the EIEIDo comprises the amino acid sequence of SEQ ID NO: 206 or 207, or an amino acid sequence having at least 70% sequence identity thereto.

39. The polynucleotide of claim 2, wherein the EIEIDo comprises a binding partner of a tetraspanin polypeptide, or a domain or fragment thereof.

40. The polynucleotide of claim 39, wherein the tetraspanin is CD63.

41. The polynucleotide of claim 39 or 40, wherein the binding partner is a human cytomegalovirus (HCMV) protein.

42. The polynucleotide of claim 41 , wherein the HCMV protein is glycoprotein M (gM).

43. The polynucleotide of claim 42, wherein the binding partner comprises the cytoplasmic tail of HCMV gM.

44. The polynucleotide of claim 43, wherein the cytoplasmic tail of HCMV gM comprises SEQ ID NO: 13 or an ammo acid sequence having at least 70% sequence identity thereto.

45. The polynucleotide of claim 2, wherein the EIEIDo comprises a Lysosome- Associated Membrane Protein 2 (LAMP2) polypeptide, or a domain or fragment thereof.

46. The polynucleotide of claim 45, wherein the EIEIDo comprises a LAMP2A polypeptide, a LAMP2B poly peptide, or a LAMP2C polypeptide.

47. The polynucleotide of claim 45 or 46, wherein the LAMP2 polypeptide comprises a human LAMP2 polypeptide or a homologous LAMP2 polypeptide from a non-human vertebrate species or an invertebrate species.

48. The polynucleotide of claim 47, wherein the LAMP2 polypeptide comprises a human LAMP2 polypeptide.

49. The polynucleotide of claim 47, wherein the LAMP2 polypeptide comprises a LAMP2 polypeptide from a non-human vertebrate species or an invertebrate species.

50. The polynucleotide of any one of claims 45-49, wherein the LAMP2 poly peptide comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence having at least 70% sequence identity thereto.

51. The polynucleotide of claim 2, wherein the EIEIDo comprises a binding partner of a LAMP2 polypeptide, or a domain or fragment thereof.

52. The polynucleotide of claim 51, wherein the binding partner comprises a motif, wherein the motif is configured to help a LAMP2 polypeptide redirect proteins to eVLPs.

53. The polynucleotide of claim 52, wherein the motif comprises the amino acid sequence KFERQ (SEQ ID NO: 17).

54. The polynucleotide of any one of claims 51-53, wherein the binding partner comprises an ExoSignal, wherein the ExoSignal is a sequence that associates with a LAMP2 protein.

55. The polynucleotide of claim 54, wherein the binding partner comprises at least 2, at least 3, at least 4, or at least 5 copies of an ExoSignal.

56. The polynucleotide of claim 54 or 55, wherein the ExoSignal comprises the amino acid sequence VKKDQAEPLHRKFERQ (SEQ ID NO: 18).

57. The polynucleotide of any one of claims 51-53, wherein the EIEIDo comprises the amino acid sequence of any one of SEQ ID NOS: 18-20. or an amino acid sequence having at least 70% sequence identity thereto.

58. The polynucleotide of claim 2, wherein the EIEIDo comprises a phosphatidylserine- binding polypeptide, or a domain or fragment thereof.

59. The polynucleotide of claim 58, wherein the phosphatidylserine-binding polypeptide comprises a lactadherin polypeptide.

60. The polynucleotide of claim 59, wherein the lactadherin polypeptide comprises a C 1C2 domain.

61. The polynucleotide of any one of claims 58-60, wherein the phosphatidylserine-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 70% sequence identity thereto.

62. The polynucleotide of claim 2, wherein the EIEIDo comprises a Platelet-Derived Growth Factor Receptor (PDGFR) polypeptide, or a domain or fragment thereof.

63. The polynucleotide of claim 62, wherein the PDGFR polypeptide, or domain or fragment thereof, comprises a human PDGFR polypeptide or a homologous PDGFR polypeptide from a non-human vertebrate species or an invertebrate species.

64. The polynucleotide of claim 63, wherein the PDGFR polypeptide comprises a human PDGFR polypeptide.

65. The polynucleotide of claim 63, wherein the PDGFR polypeptide comprises a PDGFR polypeptide from a non-human vertebrate species or an invertebrate species.

66. The polynucleotide of claim 63, wherein the homologous PDGFR polypeptide is selected from the group consisting of: Spanish mole, brushtail possum, western clawed frog, Chinese alligator, and the common sole fish.

67. The polynucleotide of any one of claims 62-66, wherein the PDGFR polypeptide, or domain or fragment thereof, comprises a transmembrane domain of a PDGFR polypeptide.

68. The polynucleotide of any one of claims 62-67, wherein the PDGFR polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 22 and 228-232, or an amino acid sequence having at least 70% sequence identity thereto.

69. The polynucleotide of claim 2, wherein the EIEIDo comprises a polypeptide capable of forming an amphipathic helix.

70. The polynucleotide of claim 69, wherein the EIEIDo is capable of inducing membrane fission by increasing membrane curvature when expressed in a cell.

71. The polynucleotide of claim 69 or 70, wherein the polypeptide capable of forming an amphipathic helix is isolated or derived from the group consisting of: M2 protein of influenza A, monoglucosyldiacylglycerol synthase (MGS) om Acholeplas ma laidlawii, septum sitedetermining protein MinD from a bacterium (e.g.. E. coll), peroxisomal membrane protein Peroxin 11 (Pexl 1 p), phospholipid N-methyltransferase PmtA from Agrobacterium tumefaciens (AtPmtA), N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acet lglucosamine transferase (MurG) from E. coli (EcMurG), EH domain-binding mitotic phosphoprotein (Epsin 1), amphiphysin from Drosophila, protein interacting with C kinase 1 (PICK1), ADP- ribosylation factor 1 (Arfl), di glucosyldiacylglycerol synthase (DGS) from Acholeplasma laidlawii, Endophilin Al, mammalian amphiphysin 2, Clathrin Assembly Lymphoid-Myeloid leukemia protein (CALM), or alpha-synuclein (a-synuclein).

72. The polynucleotide of any one of claims 69-71, wherein the polypeptide capable of forming an amphipathic helix comprises the amino acid sequence of any one of SEQ ID NOS: 23-40, or an amino acid sequence having at least 70% sequence identity thereto.

73. The polynucleotide of any one of claims 69-72, wherein the EIEIDo comprises at least two polypeptides capable of forming an amphipathic helix.

74. The polynucleotide of claim 73, wherein the at least two polypeptides are the same.

75. The polynucleotide of claim 73, wherein the at least two polypeptides are different.

76. The polynucleotide of any one of claims 73-75, wherein the at least two polypeptide capable of forming an amphipathic helix are isolated or derived from the group consisting of: M2 protein of influenza A, monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii, septum site-determining protein MinD from a bacterium (e.g., E. coll). peroxisomal membrane protein Peroxin 1 1 (Pexl Ip), phospholipid N-methyltransferase PmtA from Agrobacterium tumefaciens (AtPmtA), N-acetylmuramyl-(pentapeptide) pyrophosphoryl- undecaprenol N-acetylglucosamme transferase (MurG) from E. coli (EcMurG), EH domainbinding mitotic phosphoprotein (Epsin 1), amphiphysin from Drosophila, protein interacting with C kinase 1 (PICK1), ADP-ribosylation factor 1 (Arfl), diglucosyldiacylglycerol synthase (DGS) from Acholeplasma laidlawii, Endophilin Al, mammalian amphiphysin 2, Clathrin Assembly Lymphoid-Myeloid leukemia protein (CALM), or alpha-synuclein (a-synuclein).

77. The polynucleotide of any one of claims 73-76, wherein the at least two polypeptides are linked in-frame.

78. The polynucleotide of claim 77, wherein the at least two polypeptides are linked by a linker.

79. The polynucleotide of claim 78, wherein the linker is a glycine serine linker.

80. The polynucleotide of claim 78 or 79, wherein the linker comprises the sequence of any one of SEQ ID NOS: 209-217.

81. The polynucleotide of any one of claims 73-80, wherein the EIEIDo comprises the amino acid sequence of any one of SEQ ID NOS: 41-120, or an amino acid sequence having at least 70% sequence identity thereto.

82. The polynucleotide of any one of claims 1-81, wherein the fusion protein further comprises an endocytosis prevention motif (EPM).

83. The polynucleotide of any one of claims 1-82, wherein the antigenic polypeptide is derived from a viral protein.

84. The polynucleotide of claim 83, wherein the viral protein is derived from human metapneumo virus (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), a coronavirus, influenza, aflavivirus, or orthopoxvirus.

85. The polynucleotide of claim 84, wherein the viral protein is derived from a coronavirus, wherein the coronavirus is Middle East respiratory' syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), or SARS-CoV-2.

86. The polynucleotide of claim 85, wherein the protein is spike protein.

87. The polynucleotide of claim 84, wherein the viral protein is fusion (F) protein derived from hMPV. P1V3, or respiratory syncytial virus.

88. The polynucleotide of claim 84, wherein the viral protein is:(a) glycoprotein E (gE) derived from N7N(b) glycoprotein H (gH). glycoprotein L (gL). or glycoprotein B (gB) derived from CMV; or(c) glycoprotein C (gC) or glycoprotein D (gD) derived from HSV1 or HSV2.

89. The polynucleotide of any one of claims 1-82, wherein the antigenic polypeptide is derived from a bacterial protein.

90. The polynucleotide of claim 89, wherein the bacterial protein is derived from an acnecausing bacterium, Staphylococcus, Borrelia, E. Coli, or Chlamydia.

91. The polynucleotide of claim 90, wherein the bacterial protein is:(a) DsAl derived from Cutibcicterium acnes,'(b) esxA or esxB derived from Staphylococcus;(c) OspA derived from Borrelia. optionally Borrelia burgdorferi;(d) FimH derived from E. Coli; or(e) major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), or OmcB derived from Chlamydia.

92. The polynucleotide of any one of claims 1-82, wherein the antigenic polypeptide is derived from a parasite associated protein.

93. The polynucleotide of any one of claims 1-82, wherein the antigenic polypeptide is derived from a cancer associated protein.

94. The polynucleotide of claim 93, wherein the antigenic polypeptide is derived from a prostate cancer associated protein.

95. The polynucleotide of claim 94, wherein the prostate cancer associated protein is Six Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) or Prostate-specific membrane antigen (PS MA).

96. The polynucleotide of claim 93, wherein the antigenic polypeptide is derived from a melanoma associated protein.

97. The polynucleotide of claim 96, wherein the melanoma associated protein is Tyrosinase or transmembrane phosphatase with tensin homology7protein (TPTE).

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

99. The polynucleotide of any one of claims 1-98. wherein the polynucleotide comprises mRNA.

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

101. The polynucleotide of any one of claims 1-100, wherein the polynucleotide, or the EIEIDo or fusion protein encoded by the polynucleotide: a) allows for multivalent display of an antigenic polypeptide on an eVLP; b) allows for tailoring a lipid composition of eVLPs formed by cells expressing the fusion protein; c) enables altered modulation of eVLP surface presentation; d) enables incorporation of molecules, in addition to the antigenic polypeptide or fusion protein, into eVLPs formed by cells expressing the fusion protein; e) allows for controlling of the ratio of eVLP-associated and cell surface-associated antigens; and / or f) enables simultaneous production of a population of orthogonal eVLPs.

102. An EIEIDo comprising:(a) an envelope protein of a virus, or a domain or fragment thereof;(b) a tetraspanin polypeptide, or a domain or fragment thereof;(c) a binding partner of a tetraspanin polypeptide;(d) a Lysosome- Associated Membrane Protein 2 (LAMP2) polypeptide, or a domain or fragment thereof;(e) a binding partner of a LAMP2 polypeptide;(f) a phosphatidylserine-binding polypeptide, or a domain or fragment thereof;(g) a Platelet-Derived Growth Factor Receptor (PDGFR) polypeptide, or a domain or fragment thereof; or(h) a polypeptide comprising an amphipathic helix; wherein (a) to (h) does not comprise a naturally occurring sequence.

103. A polypeptide encoded by the polynucleotide of any one of claims 1-101.

104. A fusion protein encoded by the polynucleotide of any one of claims 1-101.

105. A fusion protein, wherein the fusion protein has an amino acid sequence comprising any one of the sequences of SEQ ID NOS: 1-207 and 218-234, or a sequence having at least 70% sequence identity thereto.

106. A cell expressing on its surface the fusion protein, or a portion of the fusion protein, of claim 104 or 105.

107. A cell expressing on its surface the antigenic polypeptide of the fusion protein, or a portion of the antigenic polypeptide of the fusion protein, of claim 104 or 105.

108. An enveloped virus-like particle (eVLP) comprising a polypeptide encoded by the polynucleotide of any one of claims 1-101 or the fusion protein of claim 104 or 105.

109. An enveloped virus-like particle (eVLP) displaying on its surface the antigenic polypeptide, or a portion of the antigenic polypeptide, of the fusion protein of claim 104 or 105.

110. The eVLP of claim 108 or 109, wherein the diameter of the eVLP ranges from about 10 nm to about 1000 nm.

111. A vector comprising the polynucleotide of any one of claims 1-101.

112. The vector of claim 11 1, wherein the vector is a viral vector.

113. The vector of claim 111, wherein the vector is a non-viral vector.

114. The vector of claim 113, wherein the non-viral vector is a plasmid.

115. The vector of claim 113, wherein the non-viral vector comprises a lipid nanoparticle (LNP).

116. The vector of claim 113, wherein the non-viral vector comprises a lipid nanoparticle (LNP) and the polynucleotide comprises mRNA.

117. 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 the polynucleotide of any one of claims 1-101, the polypeptide of claim 102, the fusion protein of claim 104 or 105, the cell of claim 106 or 107, the eVLP of any one of claims 108-110, or the vector of any one of claims 111-116.

118. A method of vaccinating a subject in need thereof, comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1-101, the polypeptide of claim 102, the fusion protein of claim 104 or 105, the cell of claim 106 or 107, the eVLP of any one of claims 108-110, or the vector of any one of claims 111-116.

119. The method of claim 117 or 118, wherein the subject has been diagnosed with an infection or is at risk of being infected by a pathogen.

120. The method of claim 119, wherein the subject has been diagnosed with an infection with a virus, a bacterium, or a parasite, or is at risk of being infected by a virus, a bacterium, or a parasite.

121. The method of claim 1 17 or 118, wherein the subject has been diagnosed with cancer or is at risk of cancer.

122. The method of any one of claims 117-121. wherein the subject is a mammalian subject.

123. The method of claim 122, wherein the subject is ahuman subject.

124. The polynucleotide of any one of claims 1-101, the polypeptide of claim 102. the fusion protein of claim 104 or 105, the cell of claim 106 or 107, the eVLP of any one of claims 108 to 110, or the vector of any one of claims 111 to 116 for use in the treatment or prevention of a disease or disorder.

125. The polynucleotide of any one of claims 1-101, the polypeptide of claim 102, the fusion protein of claim 104 or 105, the cell of claim 106 or 107, the eVLP of any one of claims 108 to 110, or the vector of any one of claims 111 to 116 for use in vaccination against a disease or disorder.

126. The polynucleotide of any one of claims 1-101, the polypeptide of claim 102, the fusion protein of claim 104 or 105, the cell of claim 106 or 107, the eVLP of any one of claims 108 to 110, or the vector of any one of claims 111 to 116 for use of claim 124 or 124, wherein the disease or disorder is an infection or a cancer.

127. The polynucleotide of any one of claims 1-101, the polypeptide of claim 102, the fusion protein of claim 104 or 105, the cell of claim 106 or 107, the eVLP of any one of claims 108 to 110, or the vector of any one of claims 111 to 116 for use of claim 126. wherein the infection is a viral infection, a bacterial infection, or a parasitic infection.

128. The polynucleotide of any one of claims 1-101, the polypeptide of claim 102. the fusion protein of claim 104 or 105, the cell of claim 106 or 107, the eVLP of any one of claims 108 to 110, or the vector of any one of claims 111 to 116 for use of claim 126, wherein the cancer is prostate cancer or melanoma129. Use of the polynucleotide of any one of claims 1-101. the polypeptide of claim 102, the fusion protein of claim 104 or 105, the cell of claim 106 or 107, the eVLP of any one of claims 108-110, or the vector of any one of claims 111-116 for manufacture of a medicament for the prevention or treatment of a disease or disorder.

130. Use of the polynucleotide of any one of claims 1-101, the polypeptide of claim 102, the fusion protein of claim 104 or 105, the cell of claim 106 or 107, the eVLP of any one of claims 108-110, or the vector of any one of claims 111-116 for manufacture of a medicament for vaccination against a disease or disorder.

131. The use of claim 129 or 130, wherein the disease or disorder is an infection or a cancer.

132. The use of claim 131, wherein the infection is a viral infection, a bacterial infection, or a parasitic infection.

133. The use of claim 131, wherein the cancer is prostate cancer or melanoma.

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