Epstein-BARR virus antigens and related uses

Abstract

Provided herein are engineered Epstein-Barr virus (EBV) polypeptides, polynucleotides encoding the same, related vectors and vaccine compositions, and related methods of making and using said compositions. The compositions disclosed herein may be useful for vaccination against EBV.

Description

EPSTEIN-BARR VIRUS ANTIGENS AND RELATED USESRELATED APPLICATIONS

[0001] This application claims the priority and benefits of U. S. Provisional Application No.63 / 714,685, filed October 31, 2024, and U. S. Provisional Application No. 63 / 789,323, filed April 15, 2024, the contents of both of which are incorporated by reference herein in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (VCCN_017_02WO_SeqList_ST26.xml; Size: 540,830 bytes; and Date of Creation: October 31, 2025) are herein incorporated by reference in their entirety.BACKGROUND

[0003] Epstein-Barr Virus (EBV) is a member of the herpes virus family that can infect human B cells and epithelial cells. EBV is known to cause infectious mononucleosis (IM) and a wide range of malignancies, including lymphoid and epithelial cancers such as Burkitt lymphoma, Hodgkin lymphoma, carcinomas like nasopharyngeal carcinoma, and gastric carcinoma, as well as multiple sclerosis. After primary infection, latent EBV persists in memory B cells and can reactivate throughout an infected individual’s life. Around half of 5-year-olds and more than 90% of adults have evidence of prior infection, but there is currently no approved vaccine. Accordingly, there is a need for interventions which can prevent and / or treat EBV infection. Provided herein are compositions and methods that address this need.SUMMARY

[0004] In one aspect, provided herein is a modified EBV glycoprotein B (EBV gB), where the modified EBV gB comprises an amino acid sequence comprising at least one modification relative to a wild type EBV gB; and optionally where the modified EBV gB exhibits an improved characteristic relative to a wild type EBV gB.

[0005] In some embodiments, the modified EBV gB is in a post-fusion conformation.

[0006] In some embodiments, the modified EBV gB is in a prefusion or prefusion-like conformation.

[0007] In some embodiments, the at least one modification relative to a wild type EBV gB comprises an amino acid substitution. In some embodiments, the modified EBV gB comprises an amino acid substitution at a position corresponding to position 609 relative to SEQ ID NO: 21.

[0008] In some embodiments, the modified EBV gB comprises an amino acid substitution of histidine (H) to glutamine (Q) at a position corresponding to position 609 relative to SEQ ID NO: 21.

[0009] In some embodiments, the modified EBV gB comprises an amino acid substitution at one or more of positions 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, R432, H316, D320, or S325, relative to SEQ ID NO: 21.

[0010] In some embodiments, the modified EBV gB comprises amino acid substitutions at positions corresponding to positions 189, L628, A175, and E634 relative to SEQ ID NO: 21.

[0011] In some embodiments, the modified EBV gB comprises substitutions at positions corresponding to positions 189, L628, A175, and E634 relative to SEQ ID NO: 21 are I89C, L628GCG, A175C, and E634C, respectively.

[0012] In some embodiments, the amino acid substitution comprises substitutions at positions corresponding to positions 189, L628, A175, E634, H316, D320, and S325 relative to SEQ ID NO: 21. In some embodiments, the substitutions at positions corresponding to positions 189, L628, A175, E634, H316, D320, and S325 relative to SEQ ID NO: 21 are I89C, L628GCG, A175C, E634C, H316I, D320Q, and S325L, respectively.

[0013] In some embodiments, the modified EBV gB comprises substitutions at positions corresponding to W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, and R432 relative to SEQ ID NO: 21.

[0014] In some embodiments, the substitutions at positions corresponding to positions W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, and R432 relative to SEQ ID NO: 21 are W112H, Y113R, W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, and R432G, respectively.

[0015] In some embodiments, the amino acid substitution comprises substitutions at positions corresponding to 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, and R432 relative to SEQ ID NO: 21.

[0016] In some embodiments, the substitutions at positions corresponding to positions 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, and R432 relative to SEQ ID NO: 21 are I89C, L628GCG, A175C, E634C, W112H, Y113R,W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, and R432G, respectively.

[0017] In some embodiments, the modified EBV gB comprises substitutions at positions corresponding to 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, R432, H316, D320, and S325 relative to SEQ ID NO: 21.

[0018] In some embodiments, the substitutions at positions corresponding to positions 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, R432, H316, D320, and S325 relative to SEQ ID NO: 21 are I89C, L628GCG, A175C, E634C, W112H, Y113R, W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, R432G, H316I, D320Q, and S325L relative to SEQ ID NO: 21, respectively.

[0019] In some embodiments, the improved characteristic is removal of a disease-associated epitope. In some embodiments, the disease-associated epitope is associated with multiple sclerosis (MS). In some embodiments, the improved characteristic is improved stability.

[0020] In some embodiments, the modified EBV gB comprises an epitope of an EBV gB, where the epitope is gB domain D-I, gB domain D-II, gB domain D-III, gB domain D-IV, or gB domain D-V. In some embodiments, the modified EBV gB comprises the sequence of SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 340, or a sequence having at least 70% sequence identity thereto. In some embodiments, the modified EBV gB comprises an amino acid substitution of at least one amino acid within a furin cleavage site.

[0021] In some embodiments, the modified EBV gB comprises an amino acid substitution of at least one amino acid within a furin cleavage site comprising the motif R-X-K / R-R.

[0022] In some embodiments, the modified EBV gB comprises an amino acid substitution of at least one amino acid within a furin cleavage site comprising the amino acid sequence of any one of SEQ ID NOS: 190-195.

[0023] In some embodiments, the modified EBV gB comprises an amino acid substitution where:

[0024] a) a native TM domain corresponding to that of a wildtype gB is substituted with a heterologous TM domain; or

[0025] b) a native TM domain and a native C-terminal domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain.

[0026] In some embodiments, the modified EBV gB comprises an amino acid substitution where:a) a native membrane proximal external region (MPER) and a native TM domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain; orb) a native membrane proximal external region (MPER), a native TM domain, and a native C-terminal domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain.

[0027] In some embodiments, the at least one modification relative to a wild type EBV gB comprises an amino acid deletion.

[0028] In certain embodiments, the modified EBV gB comprises an amino acid deletion of at least one amino acid within a furin cleavage site.

[0029] In certain embodiments, the modified EBV gB comprises an amino acid deletion of at least one amino acid within a furin cleavage site comprising the motif R-X-K / R-R.

[0030] In certain embodiments, the modified EBV gB comprises an amino acid deletion of at least one amino acid within a furin cleavage site comprising the amino acid sequence of any one of SEQ ID NOS: 190-195.

[0031] In some embodiments, the at least one modification relative to a wild type EBV gB comprises an amino acid insertion. In certain embodiments, the modified EBV gB comprises an amino acid insertion of a heterologous TM domain. In certain embodiments, the modified EBV gB an insertion of a heterologous TM domain at the C-terminus of an ectodomain of the modified EBV gB, thereby enabling expression of all or a portion thereof of the modified EBV gB on the surface of a cell in which it is expressed. In certain embodiments, the heterologous TM domain is derived from a viral protein or a bacterial protein.

[0032] In certain embodiments, the heterologous TM domain is derived from EBV gp220, influenza neuraminidase (NA), Zika virus NS2 protein, or measles virus hemagglutinin.

[0033] In certain embodiments, the heterologous TM domain comprises the amino acid sequence of any one of SEQ ID NOS: 120-125, or a sequence having at least 70% sequence identity thereto.

[0034] In certain embodiments, the modified EBV gB comprises an insertion of a linker at the C terminus, N terminus, or at the C and N terminals of the heterologous TM domain.

[0035] In some embodiments, the modified EBV gB comprises an amino acid insertion of a trimerization domain.

[0036] In certain embodiments, the trimerization domain is derived from the dimeric GCN4 protein of yeast or the foldon trimerization domain of fibritin protein of T4 bacteriophage.

[0037] In certain embodiments, the trimerization domain comprises the amino acid sequence of any one of SEQ ID NOS: 170-173, or a sequence having at least 70% sequence identity thereto.

[0038] Also disclosed herein is a modified EBV glycoprotein 350 (EBV gp350), where the modified EBV gp350 comprises an EBV gp350 amino acid sequence comprising at least one modification relative to a wild type EBV; and optionally where the modified EBV gp350 exhibits an improved characteristic relative to a wild type EBV gp350.

[0039] In some embodiments, the modified EBV gp350 comprises any one of SEQ ID NOS: 30-36, or a sequence having at least 70% sequence identity thereto.

[0040] Also disclosed herein is a modified EBV glycoprotein H (EBV gH), where the modified EBV gH comprises an EBV gH amino acid sequence comprising at least one modification relative to a wild type EBV gH; and optionally where the modified EBV gH exhibits an improved characteristic relative to a wild type EBV gH.

[0041] In some embodiments, the modified EBV gH comprises an epitope of EBV gH protein, where the epitope is gH domain D-I, gH domain D-II, gH domain D-III, or gH domain D-IV.

[0042] In some embodiments, the modified EBV gH comprises the amino acid sequence of any one of SEQ ID NOS: 1-5, or a sequence having at least 70% sequence identity thereto.

[0043] Also disclosed herein is a modified EBV glycoprotein L (EBV gL), where the modified EBV gL comprises an EBV gL amino acid sequence comprising at least one modification relative to a wild type EBV gL; and optionally where the modified EBV gL exhibits an improved characteristic relative to a wild type EBV gL.

[0044] In some embodiments, the modified EBV gL comprises the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 70% sequence identity thereto.

[0045] Also disclosed herein is a modified EBV glycoprotein 42 (EBV gp42), where the modified EBV gp42 comprises an EBV gp42 amino acid sequence comprising at least one modification relative to a wild type EBV gp42; and optionally where the modified EBV gp42 exhibits an improved characteristic relative to a wild type EBV gp42.

[0046] In some embodiments, the at least one modification comprises an amino acid substitution. In certain embodiments, the amino acid substitution is at a position corresponding to position 154 relative to SEQ ID NO: 11. In certain embodiments, the amino acid substitution is arginine (R) to lysine (K). In some embodiments, the improved characteristic is removal of a disease-associated epitope. In certain embodiments, the disease-associated epitope is associated with multiple sclerosis.

[0047] Also disclosed herein is a modified EBV glycoprotein H / glycoprotein L (EBV gH / gL), where the modified EBV gH / gL is a single chain polypeptide comprising an EBV gH amino acid sequence and an EBV gL amino acid sequence, where the modified EBVgH / gL comprises at least one modification relative to a wild type EBV gH or gL; and optionally where the modified EBV gH / gL exhibits an improved characteristic relative to a wild type EBV gH or gL.

[0048] In some embodiments, the modified EBV gH / gL comprises a modified EBV gH of the disclosure and / or a modified EBV gL of the disclosure.

[0049] Also disclosed herein is a modified EBV glycoprotein H / glycoprotein L / glycoprotein gp42 (EBV gH / gL / gp42), where the modified EBV gH / gL / gp42 is a single chain polypeptide comprising an EBV gH amino acid sequence, an EBV gL amino acid sequence, and an EBV gp42 amino acid sequence, where the modified EBV gH / gL / gp42 comprises at least one modification relative to a wild type EBV gH, gL, or gp42; and optionally where the modified EBV gH / gL / gp42 exhibits an improved characteristic relative to a wild type EBV gH, gL, or gp42.

[0050] In some embodiments, the modified EBV gH / gL / gp42 comprises a modified EBV gH of the disclosure, a modified EBV gL of the disclosure, and / or a modified EBV gp42 of the disclosure, optionally where the at least one modification relative to a wild type EBV gH, gL, or gp42 comprises an amino acid insertion of a ribosomal skip site.

[0051] Also disclosed herein is a modified EBV glycoprotein BMRF-2, where the modified EBV BMRF-2 comprises an EBV BMRF-2 amino acid sequence comprising at least one modification relative to a wild type EBV BMRF-2; and optionally where the modified EBV BMRF-2 exhibits an improved characteristic relative to a wild type EBV BMRF-2.

[0052] In some embodiments, the modified EBV BMRF-2 comprises the amino acid sequence of SEQ ID NO: 421, or a sequence having at least 70% sequence identity thereto.

[0053] Also disclosed herein is an engineered EBV polypeptide comprising at least one modified EBV glycoprotein, where the modified EBV glycoprotein comprises at least one modification relative to a wild type EBV glycoprotein, and optionally where the engineered polypeptide exhibits at least one improved characteristic.

[0054] In some embodiments, the at least one modified EBV glycoprotein comprises:

[0055] a modified EBV gB of the disclosure;

[0056] a modified EBV gp350 of the disclosure;

[0057] a modified EBV gH of the disclosure;

[0058] a modified EBV gL of the disclosure;

[0059] a modified EBV gp42 of the disclosure;

[0060] a modified EBV gH / gL of the disclosure6;

[0061] a modified EBV gH / gL / gp42 of the disclosure; or

[0062] a modified EBV BMRF-2 of the disclosure.

[0063] In some embodiments, the improved characteristic is one or more of (a) increased stability, (b) increased antigenicity, (c) increased immunogenicity, (d) increased secretion, (e) increased secretion as enveloped virus-like particles (eVLPs), (f) increased secretion as ferritin nanoparticles, (g) increased retention on the cell surface, (h) increased cell-surface expression, (i) increased exposure of target epitopes, (j) decreased exposure of off-target epitopes, (k) decreased off target immune response, (1) increased number of neutralizing epitopes targeted, (m) ability to express multiple EBV glycoproteins from a single polypeptide, (n) increased control of the rate of expression of the modified EBV glycoproteins, (o) removal of a semi-heterologous disease-associated epitope, (p) increased neutralization potency, (q) increased neutralization potency relative to total immunogenicity, (r) increased number of epitopes targeted, or a combination thereof.

[0064] In some embodiments, the at least one modification comprises an amino acid deletion, an amino acid insertion, or an amino acid substitution.

[0065] In some embodiments, the at least one modification comprises an amino acid deletion.

[0066] In certain embodiments, the amino acid deletion comprises a truncation at the C-terminal or N-terminal relative to the amino acid sequence of a wild type glycoprotein.

[0067] In some embodiments, the at least one modification comprises an amino acid substitution.

[0068] In some embodiments, the engineered EBV polypeptide comprises two or more consecutive amino acid substitutions.

[0069] In certain embodiments, the two or more consecutive amino acid substitution eliminate a furin cleavage site within the engineered EBV polypeptide.

[0070] In some embodiments, the at least one modification comprises an amino acid insertion.

[0071] In some embodiments, the amino acid insertion comprises insertion of a ribosomal skip site, an endocytosis prevention motif (EPM), a signal peptide, an endosomal sorting complex required for transport (ESCRT) recruiting domain (ERD), an ESCRT-independent eVLP inducing domain, a transmembrane (TM) domain, a trimerization domain, a ferritin sequence, a flexible linker, a cleavage site, a furin cleavage site, an EBV T-cell epitope, or a combination thereof.

[0072] In some embodiments, the engineered EBV polypeptide comprises a modified EBV gH / gL / gp42 of the disclosure, and where the amino acid insertion comprises a ribosomal skip site. In certain embodiments, the ribosomal skip site is a 2A peptide. In certain embodiments,the 2A peptide is derived from foot-and-mouth disease virus, equine rhinitis virus, porcine teschovirus-1, or Thosea asigna virus. In certain embodiments, the 2A peptide comprises the amino acid sequence of any one of SEQ ID NOS: 50-53 or a sequence having at least 70% sequence identity thereto. In certain embodiments, the ribosomal skip site is C-terminal to the gH and / or gL sequences and is N-terminal to the gp42 sequence.

[0073] In some embodiments, the amino acid insertion comprises an EPM. In certain embodiments, the EPM comprises the amino acid sequence of any one of SEQ ID NOS: 60-66 or a sequence having at least 70% sequence identity thereto.

[0074] In some embodiments, the amino acid insertion comprises an ERD, and where the ERD is:a) derived from the ESCRT and ALIX binding region (EABR) of the CEP55 protein or functional analogs from other vertebrate species;b) derived from a viral protein or portion thereof, optionally where the viral protein is derived from simian virus 5 (SV) also known as parainfluenza virus 2, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), human T-lymphotropic virus type 1 (HTLV-1), murine leukemia virus (MLV), or Mason-Pfizer monkey virus (MPMV), tick-borne encephalitis virus (TBEV), or a combination thereof; or c) an engineered ERD sequence capable of binding to at least one ESCRT protein.

[0075] In certain embodiments, the ERD is derived from an engineered ERD sequence capable of binding to at least one ESCRT protein. In certain embodiments, the at least one ESCRT protein is ALIX or TSG101. In certain embodiments, the ERD comprises the amino acid sequence of any one of SEQ ID NOS: 70-108 or a sequence having at least 70% sequence identify thereto.

[0076] In some embodiments, the amino acid insertion comprises a signal peptide. In certain embodiments, the signal peptide comprises the signal peptide from IgE, IGVH, tissue plasminogen activator (tPA), CD5, IGKV, or albumin. In certain embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOS: 40-47 or a sequence having at least 70% sequence identity thereto.

[0077] In some embodiments, the amino acid insertion comprises insertion of a transmembrane domain.

[0078] In some embodiments, insertion of the transmembrane domain:a) stabilizes the conformation of the modified EB V glycoprotein or a portion thereof; b) anchors the modified EB V glycoprotein or a portion thereof to a membrane when the polypeptide is expressed in a cell; orc) reorients the modified EBV glycoprotein, thereby increasing or decreasing exposure of at least one epitope of the glycoprotein.

[0079] In some embodiments, insertion of the transmembrane domain stabilizes the conformation of the engineered EBV polypeptide or a portion thereof.

[0080] In some embodiments, the TM domain is derived from EBV proteins, Influenza Virus proteins, synthetic TM domains, TM domains from bacteria, TM domains from viruses, and other non-human proteins with 2-pass TM domains. In some embodiments, insertion of the transmembrane domain anchors the engineered EBV polypeptide or a portion thereof to a membrane when the polypeptide is expressed in a cell. In certain embodiments, the TM domain is inserted in a position that is not naturally anchored to a membrane. In certain embodiments, the TM domain is derived from EBV gp220, influenza neuraminidase (NA), Zika virus NS2 protein, or measles virus hemagglutinin.

[0081] In some embodiments, the engineered EBV polypeptide comprises a modified EBV gB comprising a TM domain insertion, where the TM domain is inserted at the C-terminus of an ectodomain of the modified EBV gB.

[0082] In some embodiments, the engineered EBV polypeptide comprises a modified EBV gB comprising a TM domain insertion, where the TM domain is inserted at any one of the amino acid positions selected from: 1-25, 111-114, 160-162, 193-197, 545-547, and 563-565 relative to SEQ ID NO: 20.

[0083] In some embodiments, the engineered EBV polypeptide comprises the sequences of any one of SEQ ID NOs: 350-391 or a sequence having at least 70% sequence identity thereto.

[0084] In some embodiments of the engineered EBV polypeptide of the disclosure, the amino acid insertion comprises a trimerization domain. In certain embodiments, the trimerization domain is derived from yeast transcription factor GCN4 or a bacteriophage T4 fibritin foldon trimerization domain. In certain embodiments, the trimerization domain is modified. In certain embodiments, the trimerization domain comprises the amino acid sequence of any one of SEQ ID NOS: 170-173 or a sequence having at least 70% sequence identity thereto.

[0085] In some embodiments of the engineered EBV polypeptide of the disclosure, the amino acid insertion comprises a ferritin. In certain embodiments, the ferritin comprises a ferritin derived from an amphibian, an archaea, a mammal, a bacterium, a fungus, or a plant.

[0086] In certain embodiments, the ferritin comprises a ferritin derived from a bacterium. In certain embodiments, the bacterium is H. pylori, U. urealycitum, E. coli, M. tuberculosis, P. furiosus, C. tepidum, or V. cholera. In certain embodiments, the bacterium is E. coli, H.pylori, or P. furiosus. In certain embodiments, the ferritin comprises a hybrid bullfrog ferritin, where the hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from H. pylori, U. urealycitum, E. coli, M. tuberculosis, P. furiosus, C. tepidum, or V. cholera, or a portion thereof. In certain embodiments, the hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from E. coli or a portion thereof. In certain embodiments, the hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from H. pylori or a portion thereof. In certain embodiments, the ferritin comprises the sequence of any one of SEQ ID NOs: 440-452 or a sequence having at least 70% sequence identity thereto. In certain embodiments, the ferritin comprises the sequence of SEQ ID NO: 452 or a sequence having at least 70% sequence identity thereto. In certain embodiments, the ferritin comprises the sequence of SEQ ID NO: 440 or a sequence having at least 70% sequence identity thereto.

[0087] In some embodiments, the engineered EBV polypeptide comprises a peptide tag.

[0088] In some embodiments, the peptide tag is a His-tag, a Strep-tag, Flag-tag, or an Avi-tag. In certain embodiments, the peptide tag comprises at least six consecutive histidines, or the amino acid sequence of any one of SEQ ID NOS: 200-202 or a sequence with 70% sequence identity thereto.

[0089] In some embodiments, the engineered EBV polypeptide comprises a peptide linker. In certain embodiments, the peptide linker is a Gly-Ser linker. In certain embodiments, the peptide linker is derived from EBV BMRF-2, optionally where the peptide linker comprises SEQ ID NO: 180 or a sequence having at least 70% sequence identity thereto. In certain embodiments, the peptide linker is derived from EBV gp350 or bullfrog ferritin. In certain embodiments, the peptide linker is flanked by one, two, or three amino acids on the N-terminus, the C-terminus, or both the N and C terminals. In certain embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOS: 180-188 or a sequence having at least 70% sequence identity thereto.

[0090] In some embodiments, the engineered EBV polypeptides of the disclosure comprise the amino acid sequence of any one of SEQ ID NOS: 210-231, 240-250, 270-283, 290-293, 300-331, 340-391, 400-409, 420-421, and 430-433 or a sequence having at least 70% sequence identity thereto.

[0091] In some embodiments, the engineered EBV polypeptides of the disclosure comprise the sequence of any one of SEQ ID NOS: 34, 328, and 388, or a sequence having at least 70% sequence identity thereto.

[0092] In some embodiments, the engineered EBV polypeptides of the disclosure comprise a modified EBV gH / gL / gp42.

[0093] In some embodiments, the engineered EBV polypeptides of the disclosure comprise an amino acid insertion, where the amino acid insertion comprises a signal peptide, a, linker, a ribosomal skip site, a TM domain, an EPM, an ERD, a furin cleavage site, a ferritin, or a combination thereof.

[0094] In some embodiments, the engineered EBV polypeptide comprises, from N terminus to C terminus:a signal peptide, EBV gL, a linker, EBV gH, a TM domain, a ribosomal skip site, and EBV gp42;a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an ERD, a ribosomal skip site, and EBV gp42;a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an EPM, an ERD, a ribosomal skip site, and EBV gp42;a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an ERD, a furin cleavage site, a ribosomal skip site, and EBV gp42;a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an EPM, an ERD, a furin cleavage site, a ribosomal skip site, and EBV gp42;a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, and a TM domain; a signal peptide, EBV gp42, a linker, EBV gL, a linker, and EBV gH;a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, a TM domain, an EPM, and an ERD;a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, an EPM, and an ERD;a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, a TM domain, and an ERD; ora signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, a ferritin.

[0095] In some embodiments, the linker is a peptide linker derived from EBV BMRF-2 or EBV gp350 or a Gly-Ser linker. In certain embodiments, the amino acid sequence of the linker comprises any one of SEQ ID NO: 180-188 or a sequence having at least 70% sequence identity thereto.

[0096] In some embodiments, the ribosomal skip site is a 2A peptide. In some embodiments, the ribosomal skip site comprises the amino acid sequence of any one of SEQ ID NOS: 50-53 or a sequence having at least 70% sequence identity thereto.

[0097] In some embodiments, the EPM comprises the amino acid sequence of any one of SEQ ID NOS: 60-66 or a sequence having at least 70% sequence identity thereto. In some embodiments, the TM domain is a single-pass TM domain, a TM domain derived from EBV gp350, or the native TM domain from EBV gH.

[0098] In some embodiments, the ERD is derived from a viral protein. In some embodiments, the viral protein is derived from MPMV, HIV, or a combination thereof, optionally where the amino acid sequence of the ERD comprises SEQ ID NO: 37 or SEQ ID NO: 38 or a sequence with 70% sequence identity thereto.

[0099] Also disclosed herein is an enveloped virus-like particle (eVLP) comprising one or more of: a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; or an engineered EBV polypeptide of the disclosure.

[0100] Also disclosed herein is an enveloped virus-like particle (eVLP) displaying on its surface all or a portion thereof of one or more of: a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; or an engineered EBV polypeptide of the disclosure.

[0101] Also disclosed herein is a cell comprising one or more of: a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; or an engineered EBV polypeptide of the disclosure.

[0102] Also disclosed herein is a cell displaying on its surface all or a portion thereof of: a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; or an engineered EBV polypeptide of the disclosure.

[0103] Also disclosed herein is a polynucleotide encoding the amino acid sequence of: a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of thedisclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; or an engineered EBV polypeptide of the disclosure.

[0104] In some embodiments, the polynucleotide comprises DNA.

[0105] In some embodiments, the polynucleotide comprises RNA.

[0106] In some embodiments, the RNA comprises mRNA, self-amplifying RNA, transamplifying RNA, or circular RNA.

[0107] In some embodiments, the RNA comprises modified nucleosides.

[0108] In some embodiments, the polynucleotide comprises an Internal Ribosomal Entry Site (IRES) sequence. In certain embodiments, the IRES sequence comprises the sequence of SEQ ID NO: 182 or a sequence having at least 70% sequence identity thereto.

[0109] In some embodiments, the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NOS: 470-493 or a sequence having at least 70% sequence identity thereto.

[0110] Also disclosed herein is a vector comprising one or more of a polynucleotide of the disclosure. In some embodiments, the vector is a viral vector.[OHl] In certain embodiments, the viral vector is: an adenovirus; an Adeno-associated virus (AAV); a vesiculovirus; a retrovirus; a herpesvirus; or a vaccinia virus.

[0112] In certain embodiments, the viral vector is an adenovirus and where the adenovirus is a human adenovirus selected from the group consisting of: Adenovirus 3, Adenovirus 5, Adenovirus 26, Adenovirus 35, and Adenovirus 48.

[0113] In certain embodiments, the viral vector is a non-human adenovirus, optionally where the non-human adenovirus is a primate-derived adenovirus. In certain embodiments, the non-human adenovirus is a chimpanzee adenovirus. In certain embodiments, the viral vector is a vesiculovirus and where the vesiculovirus is Vesicular Stomatitis Virus (VSV).

[0114] In certain embodiments, the viral vector is a retrovirus and where the retrovirus is a lentivirus. In certain embodiments, the viral vector is a herpesvirus and where the herpesvirus is cytomegalovirus. In certain embodiments, the viral vector is a vaccinia virus and where the vaccinia virus is modified vaccinia Ankara (MV A).

[0115] In some embodiments, the vector is a non-viral vector. In certain embodiments, the non-viral vector is a plasmid. In certain embodiments, the non-viral vector is a lipid nanoparticle (LNP).

[0116] Also disclosed herein is a vaccine composition comprising: a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBVgH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; an engineered EBV polypeptide of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; or a vector of the disclosure.

[0117] In some embodiments, the vaccine composition comprises about O.lug to about lOOug of the engineered EBV polypeptide of the disclosure; or about 0. lug to about lOOug of the polynucleotide of the disclosure.

[0118] In some embodiments, the vaccine composition comprises at least two different engineered EBV polypeptides of the disclosure; at least two different polynucleotides of the disclosure, optionally where the at least two different polynucleotides are encapsulated in separate LNPs; or at least two different vectors of the disclosure.

[0119] In some embodiments, the vaccine composition comprises at least a first and a second engineered EBV polypeptide of the disclosure, where the first polypeptide comprises a first ferritin and the second polypeptide comprises a second ferritin, and where the first and the second ferritin are not the same; or at least a first and a second polynucleotide of the disclosure, where the first polynucleotide encodes a first polypeptide comprising a first ferritin and the second polynucleotide encodes a second polypeptide comprising a second ferritin, and where the first and the second ferritin are not the same.

[0120] In certain embodiments, the first and the second ferritins are derived from a ferritin of an amphibian, a bacterium, a fungus, an archaea, a mammal, or a plant.

[0121] In certain embodiments, the bacterium is selected from the group consisting of: H. Pylori, U. Urealycitum, E. Coli, M. Tuberculosis, P. Furiosus, C. Tepidum, and V. Cholera.

[0122] In certain embodiments, the amphibian is a bullfrog.

[0123] In certain embodiments, the first or the second ferritin comprises a hybrid bullfrog ferritin, where the hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from H. pylori, U. urealycitum, E. coli, M. tuberculosis, P. furiosus, C. tepidum, or V. cholera, or a portion thereof.

[0124] In some embodiments, the vaccine composition comprises a first and a second engineered EBV polypeptides of the disclosure, where:the first engineered EBV polypeptide comprises gp350; andthe second engineered EBV polypeptide comprises:modified EBV gH and gL; modified EBV gH, gL, and gp42, or modified EBV gH, gL, and gp42; and optionally a linker derived from EBV gp350 or EBV BMRF-2 or a Gly-Ser linker.

[0125] In some embodiments, the vaccine composition comprises a first, a second, and a third engineered EBV polypeptides of the disclosure, where:the first engineered EBV polypeptide comprises a modified EBV gB;the second engineered EBV polypeptide comprises gp350; andthe third engineered EBV polypeptide comprises:modified EBV gH and gL; modified EBV gH, gL, and gp42, or modified EBV gH, gL, and gp42; and optionally a linker derived from EBV gp350 or EBV BMRF-2 or a Gly-Ser linker.

[0126] In some embodiments, the vaccine composition comprises a first, a second, and a third polynucleotide of the disclosure, where:the first polynucleotide encodes a first engineered EBV polypeptide comprising a modified EBV gB;the second polynucleotide encodes a second engineered EBV polypeptide comprising a modified EBV gp350; andthe third polynucleotide encodes a third engineered EBV polypeptide comprising: modified EBV gH and gL; modified EBV gH, gL, and gp42; or modified EBV gH, gL, and gp42 containing a linker derived from EBV gp350 or EBV BMRF-2 or a gly-ser linker.

[0127] In certain embodiments, a glycoprotein or a portion thereof of the first polypeptide is expressed on the surface of an eVLP; a glycoprotein or a portion thereof of the first polypeptide and a glycoprotein of the third polypeptide are expressed on the surface of eVLPs; or a glycoprotein or a portion thereof of the first polypeptide, a glycoprotein of the second polypeptide, and a glycoprotein of the third polypeptide are expressed on the surface of eVLPs.

[0128] In certain embodiments, the first engineered EBV polypeptide comprises a first ferritin and the third engineered EBV polypeptide comprises a second ferritin, and where the first and the second ferritin are not the same.

[0129] In certain embodiments, the first and / or the second ferritin comprises a ferritin derived from an amphibian, an archaea, a mammal, a bacterium, a fungus, or a plant.

[0130] In certain embodiments, the first and / or the second ferritin comprises a ferritin derived from a bacterium, optionally where the bacterium is H. pylori, U. urealycitum, E. coli, M. tuberculosis, P. furiosus, C. tepidum, or V. cholera.

[0131] In certain embodiments, the first and / or the second ferritin comprises a ferritin derived from an amphibian, optionally where the amphibian is a bullfrog.

[0132] In certain embodiments, the first and / or the second ferritin comprises a hybrid bullfrog ferritin, where the hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from H. pylori, U. urealycitum, E. coli, M. tuberculosis, P. furiosus, C. tepidum, or V. cholera, or a portion thereof.

[0133] In certain embodiments, the second engineered EBV polypeptide comprises a third ferritin and where the first, the second, and the third ferritin are not the same.

[0134] In some embodiments, the vaccine composition comprises a fourth engineered EBV polypeptide comprising a modified EBV BMRF-2 protein.

[0135] In some embodiments, the vaccine composition comprises a fourth polynucleotide encoding a fourth engineered EBV polypeptide comprising a modified EBV BMRF-2 protein. In certain embodiments, the fourth engineered EBV polypeptide comprises an ERD or ESCRT -independent eVLP inducing domain, and is expressed on the surface of a fourth eVLP. In certain embodiments, the fourth engineered EBV polypeptide comprises a fourth ferritin, and where the first, the second, the third, and the fourth ferritins are not the same.

[0136] In certain embodiments, the polynucleotides comprise mRNA, self-amplifying RNA, trans-amplifying RNA, or circular RNA.

[0137] In some embodiments, the vaccine composition comprises at least one adjuvant.

[0138] In certain embodiments, the adjuvant comprises CpG, alum, Alhydrogel, QS-21, saponin, MPLA, squalene, Adju-Phos, a TLR agonist, or a combination thereof. In certain embodiments, the TLR agonist is an agonist of TLR7 and / or TLR8.

[0139] In some embodiments, the vaccine composition comprises one or more pharmaceutically acceptable carrier, excipient, or diluent.

[0140] Also disclosed herein is a kit comprising: a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; an engineered EBV polypeptide of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; or a vector of the disclosure; or a vaccine composition of the disclosure; and instructions for use.

[0141] Also disclosed herein is a method of stabilizing EBV gB protein in a prefusion like conformation, where the method comprises inserting a heterologous transmembrane domain into the gB protein, whereby the TM domain anchors gB in a prefusion like conformation.

[0142] Also disclosed herein is a method of expressing an EBV gB protein on the surface of a membrane, where the method comprises inserting a heterologous TM domain into one or more sites in the gB protein.

[0143] In certain embodiments, the TM domain is inserted in anchor site 1, 2, 3, 4, 5, or 6 of gB. In certain embodiments, the TM domain is inserted in a fusion loop of gB.

[0144] In certain embodiments, the TM domain is inserted in a fusion loop of gB comprising an amino acid sequence of GWYA (SEQ ID NO: 183) or WLIWT (SEQ ID NO: 184).

[0145] In certain embodiments, the TM domain is a two-pass TM domain.

[0146] In certain embodiments, the two-pass transmembrane domain is derived from Zika virus NS2 protein or influenza virus NA protein.

[0147] In some embodiments, the method of expressing an EBV gB protein on the surface of a membrane comprises inserting a linker into the gB protein. In certain embodiments, the linker is a flexible linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker is a Gly-Ser linker. In certain embodiments, linker is directly connected to the N terminus or C terminus of the TM domain. In certain embodiments, the length of the linker corresponds to the distance of the insertion site from the membrane.

[0148] In certain embodiments, the length of the linker is between 1 and 40 amino acids.

[0149] Also disclosed herein is a method of treating an EBV associated cancer or a symptom thereof in a subject having an EBV associated cancer, comprising administering to the subject an effective amount of a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; an engineered EBV polypeptide of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; or a vector of the disclosure; or a vaccine composition of the disclosure.

[0150] Also disclosed herein is a method of inducing anti-EBV antibodies and / or inducing a T cell response against EBV in a subject, comprising administering to the subject an effective amount of a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; an engineered EBV polypeptide of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; or a vector of the disclosure; or a vaccine composition of the disclosure.

[0151] In some embodiments, the anti-EBV antibodies bind at least one epitope of EBV gB, gp350, gH, gL, gp42, or BMRF-2. In some embodiments, the antibodies bind to EBV gB domain D-II or gB domain D-IV.

[0152] Also disclosed herein is a method for inducing an EBV-specific immune response, comprising administering to the subject an effective amount of a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; an engineered EBV polypeptide of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; a vector of the disclosure; or a vaccine composition of the disclosure.

[0153] In some embodiments, the subject is a human. In some embodiments, the subject is: immunosuppressed or immunocompromised; an adolescent; or under the age of 18.

[0154] In some embodiments, the subject has had a transplant or will have a transplant.

[0155] In some embodiments, the subject has a prior EBV infection.

[0156] In some embodiments, the subject does not have a prior EBV infection.

[0157] In some embodiments, the route of administration is intramuscular.

[0158] Also disclosed herein is an use of a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; an engineered EBV polypeptide of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; a vector of the disclosure; or a vaccine composition of the disclosure for manufacture of a medicament for the prevention or treatment of EBV infection or an EBV-associated disease or disorder.

[0159] Also disclosed herein is a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; an engineered EBV polypeptide of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; a vector of the disclosure; or a vaccine composition of the disclosure for manufacture of a medicament for vaccination against EBV infection.

[0160] Also disclosed herein is a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure;a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; an engineered EBV polypeptide of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; a vector of the disclosure; or a vaccine composition of the disclosure for use in the treatment or prevention of EBV infection or an EBV-associated disease or disorder.

[0161] Also disclosed herein is a modified EBV gB of the disclosure; a modified EBV gp350 of the disclosure; a modified EBV gH of the disclosure; a modified EBV gL of the disclosure; a modified EBV gp42 of the disclosure; a modified EBV gH / gL of the disclosure; a modified EBV gH / gL / gp42 of the disclosure; a modified EBV BMRF-2 of the disclosure; an engineered EBV polypeptide of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; a vector of the disclosure; or a vaccine composition of the disclosure for use in the vaccination against EBV infection.BRIEF DESCRIPTION OF THE DRAWINGS

[0162] FIG. 1A provides a schematic of EBV showing four protein complexes that can be targeted for vaccine generation, including (1) gp350, (2) a heterotrimeric protein complex made up of gH, gL, and gp42, (3) a homotrimeric protein complex of gB, and (4) BMRF-2, and the proteins’ interaction with target B cells or epithelial cells.

[0163] FIG. IB provides a schematic representation of an exemplary gB ferritin nanoparticle: the depiction is of a post fusion gB trimer arrayed on a surface of a ferritin nanoparticle.

[0164] FIG. 2 provides a schematic of a crystal structure of an exemplary gB ferritin nanoparticle.

[0165] FIG. 3 provides a graph showing the binding of gB ferritin nanoparticles in the supernatant of mammalian cells.

[0166] FIG. 4 provides a representative image of an SDS-PAGE gel of supernatants containing gB ferritin nanoparticles.

[0167] FIG. 5 provides a representative image of an SDS-PAGE gel of a gH / gL / gp42 ferritin nanoparticle and a gB ferritin nanoparticle.

[0168] FIG. 6A provides a graph of dynamic light scattering (DLS) data of a gB ferritin nanoparticle.

[0169] FIG. 6B provides a graph of DLS data of a gp350 ferritin nanoparticle.

[0170] FIG. 6C provides a graph of DLS data of a gH / gL / gp42 ferritin nanoparticle.

[0171] FIG. 7 provides a graph of differential scanning fluorimetry data of a gB ferritin nanoparticle.

[0172] FIG. 8 provides a graph of biolayer interferometry (BLI) binding of a gB ferritin nanoparticle.

[0173] FIG. 9A provides a graph of an ELISA of B cell neutralization using sera from mice administered vaccine combinations of EBV ferritin nanoparticles. Each dot indicates the neutralizing potency of sera from a single mouse.

[0174] FIG. 9B provides a graph of an ELISA of epithelial cell neutralization using sera from mice administered vaccine combinations of EBV ferritin nanoparticles. Each dot indicates the neutralizing potency of sera from a single mouse.

[0175] FIGS. 10A-10C provides graphs of sera binding by ELISAs from mice immunized with various vaccine combinations of EBV ferritin nanoparticles.

[0176] FIG. 11 provides an SDS PAGE gel showing the purified forms of 6 different gHgLgp42-ferritin variants. The molecular weight of the ladder is shown on the left.

[0177] FIG. 12 provides a Western blot using a gHgLgp42 directed antibody showing that the bands seen in the SDS page gel in FIG. 11 are the anticipated proteins.

[0178] FIG. 13 provides DLS curves demonstrating the relative differences in size between the gHgLgp42-ferritin variants.

[0179] FIG. 14 provides sera B cell neutralization data from mice immunized with combination protein vaccines containing combinations of gp350, gHgLgp42, and gB displayed on ferritin nanoparticles and formulated with alum adjuvant. Mice were immunized with the indicated proteins at day 0 and day 21, and neutralizing potency of sera were assayed at day 28. Each dot indicates the neutralizing potency represented as IC50 of sera from a single mouse.

[0180] FIG. 15 provides a graph of sera epithelial cell neutralization data from mice immunized with combination protein vaccines containing combinations of gp350, gHgLgp42, and gB displayed on ferritin nanoparticles and formulated with alum adjuvant.

[0181] FIG. 16 provides an SDS PAGE gel depicting purified protein forms of gp350 variants, with NR referring to non-reducing and R referring to reducing. A molecular weight ladder and kDa molecular weights are shown on left.

[0182] FIG. 17 provides graphs of biolayer interferometry binding curves for the gp350 variants as shown in FIG. 16.

[0183] FIG. 18 provides a schematic of the antigen designs used in recombinant protein vaccines along with pictorial representation of the predicted structures of the antigen designs and how they may be combined into multicomponent vaccines.

[0184] FIG. 19 provides a graph showing day 28 sera B cell neutralization data from mice immunized at day 0 and day 21 with recombinant protein vaccines containing subunit proteins or proteins displayed on ferritin nanoparticles. Each dot indicates the neutralizing potency represented as IC50 of sera from a single mouse.

[0185] FIG. 20 provides a graph showing day 28 sera epithelial cell neutralization from mice immunized at day 0 and day 21 with recombinant protein vaccines containing subunit proteins or proteins displayed on ferritin nanoparticles. Each dot indicates the neutralizing potency represented as IC50 of sera from a single mouse. (+)- and (-)-sera refer to neutralizing responses against human serum control samples which were known to be positive or negative for prior EBV infection.

[0186] FIG. 21 provides a schematic of the antigen designs used in mRNA vaccines encoding cell anchored proteins and how they may be combined into multicomponent vaccines.

[0187] FIG. 22 provides a schematic of the antigen designs used in mRNA vaccines encoding proteins containing an endocytosis prevention motif (EPM) and ESCRT-recruiting domain (ERD) motif. The ERD utilized in the figure is ESCRT- and ALIX-binding region (EABR).

[0188] FIG. 23 provides a schematic of the antigen designs used in mRNA vaccines encoding proteins containing an orthogonal ferritin motif.

[0189] FIG. 24 provides a Western blot using a gp220 directed antibody showing protein expression following transfection with selected mRNA constructs used for vaccination in FIGs 27-29

[0190] FIG. 25 provides a Western blot using a gH / gL / gp42 directed antibody showing protein expression following transfection with selected mRNA constructs used for vaccination in FIGs 27-29.

[0191] FIG. 26 provides a Western blot showing protein expression following transfection with selected mRNA constructs used for vaccination in FIGs 27-29.

[0192] FIG. 27 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp350) conjugated beads as quantified via Luminex at day 36 following immunization on day 0 with the indicated constructs delivered as mRNA. Each dot indicates the antibody binding of sera from a single mouse to DI 23.

[0193] FIG. 27B provides a graph showing sera binding to D123 (the receptor binding domain of gp350) conjugated beads as quantified via Luminex at day 56 or 64 followingimmunizations on day 0 and 36 with the indicated constructs delivered as mRNA. Each dot indicates the antibody binding of sera from a single mouse to DI 23.

[0194] FIG. 27C provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex at day 36 following immunization on day 0 with the indicated constructs delivered as mRNA. Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0195] FIG. 27D provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex at day 56 or 64 following immunizations on day 0 and 36 with the indicated constructs delivered as mRNA. Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0196] FIG. 27E provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex at day 36 following immunization on day 0 with the indicated constructs delivered as mRNA. Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0197] FIG. 27F provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex at day 56 or 64 following immunizations on day 0 and 36 with the indicated constructs delivered as mRNA. Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0198] FIG. 27G provides a graph showing sera binding to gB conjugated beads as quantified via Luminex at day 36 following immunization on day 0 with the indicated constructs delivered as mRNA. Each dot indicates the antibody binding of sera from a single mouse to gB.

[0199] FIG. 27H provides a graph showing sera binding to gB conjugated beads as quantified via Luminex at day 56 or 64 following immunizations on day 0 and 36 with the indicated constructs delivered as mRNA. Each dot indicates the antibody binding of sera from a single mouse to gB.

[0200] FIG. 28 provides a graph showing B cell neutralizing potency of day 56 or day 64 sera in the 4E3 cell line, as analyzed from mice immunized at day 0 and day 36 with mRNA encoding the indicated antigens. Each dot indicates the neutralizing potency of sera from a single mouse.

[0201] FIG. 29 provides a graph showing day 56 or day 64 sera epithelial cell neutralization in the HEK-293T cell line, as analyzed from mice immunized at day 0 and day 36 with mRNA encoding the indicated antigens. Each dot indicates the neutralizing potency of sera from a single mouse.

[0202] FIG. 30 provides a transmission electron microscopy (TEM) image of enveloped virus like particles (eVLPs) purified from expi293 cells transfected with a plasmid encoding gB-FP-Mut-EPM-EABR with an N-terminal strep tag.

[0203] FIG. 31 provides a Western blot showing gp220 expression, as detected by a monoclonal antibody directed against gp220, 72A1, in the supernatant and lysate following transfection of HEK-293T cells with the indicated gp350-E. coli ferritin mRNA, delivered either with a lipofection reagent or encapsulated in an LNP.

[0204] FIG. 32 provides a Western blot showing expression in the supernatant and lysate following transfection of HEK-293T cells with the indicated gHgLgp42-H. Pylori ferritin mRNA, delivered either with a lipofection reagent or encapsulated in an LNP. Expression was detected by a mixture of two monoclonal antibodies: CL59 is directed against gH domain 3 while 5E3 is directed against gp42.

[0205] FIG. 33 provides representative TEM images of gB ferritin constructs containing the indicated ferritins and expressed in expi293 cells and purified using size exclusion chromatography.

[0206] FIG. 34 provides a graph showing comparative in-vitro potency of three gB ferritin mRNA designs. Antigen expression on HeLa cells that were treated with LNPs containing each of the mRNA designs was detected by a monoclonal antibody (3 A5) directed against gB Domain IV, measured by flow cytometry, and plotted as the area under the curve (AUC) relative to an untreated control.

[0207] FIG. 35 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp350) conjugated beads as quantified via Luminex at day 20, following immunization on day 0 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp350.

[0208] FIG. 35B provides a graph showing sera binding to DI 23 (the receptor binding domain of gp350) conjugated beads as quantified via Luminex at day 34, following immunization on days 0 and 20 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp350.

[0209] FIG. 35C provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex at day 20, following immunization on day 0 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0210] FIG. 35D provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads s quantified via Luminex at day 34, following immunization on days 0 and 20 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0211] FIG. 35E provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex at day 20, following immunization on day 0 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0212] FIG. 35F provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex at day 34, following immunization on days 0 and 20 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0213] FIG. 35G provides a graph showing sera binding to gp42 conjugated beads as quantified via Luminex at day 20, following immunization on day 0 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp42.

[0214] FIG. 35H provides a graph showing sera binding to gp42 conjugated beads as quantified via Luminex at day 34, following immunization on days 0 and 20 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp42.

[0215] FIG. 351 provides a graph showing sera binding to gB conjugated beads as quantified via Luminex at day 20, following immunization on day 0 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB.

[0216] FIG. 35J provides a graph showing sera binding to gB conjugated beads as quantified via Luminex at day 34 following immunization on days 0 and 20 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB.

[0217] FIG. 35K provides a graph showing sera binding to gB Domain II conjugated beads as quantified via Luminex at day 20 following immunization on day 0 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain II.

[0218] FIG. 35L provides a graph showing sera binding to gB Domain II conjugated beads as quantified via Luminex at day 34 following immunization on days 0 and 20 with theindicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain II.

[0219] FIG. 35M provides a graph showing sera binding to gB Domain IV conjugated beads as quantified via Luminex at day 20, following immunization on day 0 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain IV.

[0220] FIG. 35N provides a graph showing sera binding to gB Domain IV conjugated beads as quantified via Luminex at day 34, following immunization on days 0 and 20 with the indicated ferritin nanoparticle constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain IV.

[0221] FIG. 36 provides a graph showing the neutralizing potency of sera collected at day 34 from mice immunized with mRNA encoding the indicated ferritin nanoparticles (2.0 or 0.4 pg per antigen) at day 0 and day 20. Each dot indicates the neutralizing potency of sera from a single mouse.

[0222] FIG. 37 provides a cartoon depicting improved gp350 antigen designs, with three different stalk lengths and an endocytosis prevention motif (EPM) followed by an ESCRT-recruiting domain (ERD) affixed to the C-terminus.

[0223] FIG. 38 provides a graph showing in vitro potency of gp350 antigen designs. Antigen expression on HeLa cells that were treated with LNPs containing each of the mRNA designs was detected by a monoclonal antibody (72A1) directed against gp220, measured by flow cytometry, and plotted as the area under the curve (AUC) relative to an untreated control.

[0224] FIG. 39 provides a cartoon depicting gH / gL / gp42 antigen designs.

[0225] FIG. 40 provides a graph showing in vitro potency of gH / gL / gp42 antigen designs. Antigen expression on HeLa cells that were treated with each of the mRNA designs was detected by a monoclonal antibody (CL59) directed against gH domain 3, measured by flow cytometry, and plotted as the area under the curve (AUC) relative to an untreated control.

[0226] FIG. 41 provides a cartoon depicting gB antigen designs.

[0227] FIG. 42 provides a Western blot showing expression of various transembrane (TM)-anchored prefusion-like gB antigens in the supernatant of expi293 cells 3 days posttransfection with six plasmid constructs containing TM anchor sites at various locations and an EPM-EABR motif for secretion as eVLPs. gB-FP-mut-EPM-EABR was used as a control.

[0228] FIG. 43 provides in vitro potency of gB antigen designs toward 3A3, an antibody directed against the gB domain D-II epitope. Cell surface antigen expression on HeLa cells that were treated that were treated with LNPs containing each of the mRNA designs wasdetected using a monoclonal antibody (3 A3) directed against gB domain II, measured by flow cytometry, and plotted as the area under the curve (AUC) relative to an untreated control.

[0229] FIG. 44 provides in vitro potency of gB antigen designs toward 3 A5, an antibody directed against the gB domain D-IV epitope. Cell surface antigen expression on HeLa cells that were treated with LNPs containing each of the indicated RNAs was detected using a monoclonal antibody (3 A5) directed against gB domain IV, measured by flow cytometry, and plotted as the area under the curve (AUC) relative to an untreated control.

[0230] FIG. 45 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp350) conjugated beads as quantified via Luminex. Sera was collected on day 20 from mice immunized on day 0 with the indicated constructs delivered as mRNA (.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp350.

[0231] FIG. 45B provides a graph showing sera binding to D123 (the receptor binding domain of gp350) conjugated beads as quantified via Luminex. Sera was collected on day 34 from mice immunized on days 0 and 20 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp350.

[0232] FIG. 45C provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 20 from mice immunized on day 0 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0233] FIG. 45D provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 34 from mice immunized on days 0 and 20 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse togH / gL / gp42.

[0234] FIG. 45E provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 20 from mice immunized on day 0 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0235] FIG. 45F provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 34 from mice immunized on days 0 and 20 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0236] FIG. 45G provides a graph showing sera binding to gp42 conjugated beads as quantified via Luminex. Sera was collected on day 20 from mice immunized on day 0 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp42.

[0237] FIG. 45H provides a graph showing sera binding to gp42 conjugated beads as quantified via Luminex. Sera was collected on day 34 from mice immunized on days 0 and 20 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp42.

[0238] FIG. 451 provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 20 from mice immunized on day 0 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB.

[0239] FIG. 45J provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 34 from mice immunized on days 0 and 20 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB.

[0240] FIG. 45K provides a graph showing sera binding to gB Domain II conjugated beads as quantified via Luminex. Sera was collected on day 20 from mice immunized on day 0 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain II.

[0241] FIG. 45L provides a graph showing sera binding to gB Domain II conjugated beads as quantified via Luminex. Sera was collected on day 34 from mice immunized on days 0 and 20 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain II.

[0242] FIG. 45M provides a graph showing sera binding to gB Domain IV conjugated beads as quantified via Luminex. Sera was collected on day 20 from mice immunized on day 0 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain IV.

[0243] FIG. 45N provides a graph showing sera binding to gB Domain IV conjugated beads as quantified via Luminex. Sera was collected on day 34 from mice immunized on days 0 and 20 with the indicated constructs delivered as mRNA (2.0 or 0.4 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain IV.

[0244] FIG. 46 provides a graph showing B cell neutralization in the 4E3 cell line by sera collected on day 34 from mice immunized on day 0 and day 20 with mRNA encoding the indicated antigens. Each dot indicates the neutralizing potency of sera from a single mouse.

[0245] FIG. 47 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp350) conjugated beads as quantified via Luminex. Sera was collected on day 21 from mice immunized on day 0 with the indicated multiantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp350.

[0246] FIG. 47B provides a graph showing sera binding to DI 23 (the receptor binding domain of gp350) conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated mulitantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp350.

[0247] FIG. 47C provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 21 from mice immunized on day 0 with the indicated multiantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse togH / gL / gp42.

[0248] FIG. 47D provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulation delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0249] FIG. 47E provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 21 from mice immunized on day 0 with the indicated mulitantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0250] FIG. 47F provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0251] FIG. 47G provides a graph showing sera binding to gp42 conjugated beads as quantified via Luminex. Sera was collected on day 21 from mice immunized on day 0 withthe indicated multiantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp42.

[0252] FIG. 47H provides a graph showing sera binding to gp42 conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulation delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gp42.

[0253] FIG. 471 provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 21 from mice immunized on day 0 with the indicated multiantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB.

[0254] FIG. 47J provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB.

[0255] FIG. 47K provides a graph showing sera binding to gB Domain II conjugated beads as quantified via Luminex. Sera was collected on day 21 from mice immunized on day 0 with the indicated multiantigen formulation delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain II.

[0256] FIG. 47L provides a graph showing sera binding to gB Domain II conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain II.

[0257] FIG. 47M provides a graph showing sera binding to gB Domain IV conjugated beads as quantified via Luminex. Sera was collected on day 21 from mice immunized on day 0 with the indicated multiantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain IV.

[0258] FIG. 47N provides a graph showing sera binding to gB Domain IV conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (1 or 0.2 pg per antigen). Each dot indicates the antibody binding of sera from a single mouse to gB Domain IV.

[0259] FIG. 48 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp350) conjugated beads as quantified via Luminex. Sera was collected on days 0 and 29 from nonhuman primates (NHPs) immunized on day 0 with the indicated vaccines.Each dot indicates the antibody binding of sera from a single nonhuman primate (NHP) to gp350.

[0260] FIG. 48B provides a graph showing sera binding to gH / gL / gp42 conjugated beads as quantified via Luminex. Sera was collected on days 0 and 29 from NHPs immunized on day 0 with the indicated vaccines. Each dot indicates the antibody binding of sera from a single NHP to the given bead.

[0261] FIG. 49 provides a graph showing sera B cell neutralization in the 4E3 cell line by sera collected on days 0 and 29 from NHPs immunized with the indicated vaccines on day 0. Each dot indicates the neutralizing potency of sera from a single NHP, and connecting lines indicate trends for a single animal.

[0262] FIG. 50 provides a graph showing sera epithelial cell neutralization in the HEK-293T cell line by sera collected on days 0 and 29 from select groups of NHPs immunized with mRNA encoding the indicated antigens on day 0. Each dot indicates the neutralizing potency of sera from a single NHP, and connecting lines indicate trends for a single animal.

[0263] FIG. 51 provides a schematic depicting various EBV gB antigen design strategies including prefusion gB stabilizing mutations, fusion loop mutation strategies, transmembrane domains, trimerization motifs, and domains for eVLP formation.

[0264] FIG. 52 provides graphs showing cell surface and total expression, as measured by flow cytometry and plotted as MFI, of gB antigen designs. Expi293 cells were treated with mRNA encoding the indicated antigen designs and collected after 24 hours to evaluate cell surface expression, fixed as a control, or fixed and permeabilized to detect total expression. Antigen expression was detected using two monoclonal antibodies, AMM05 human Fc and AMM03 mouse Fc, and corresponding secondary antibodies, goat anti-human IgG PE (top panel) and goat anti-mouse IgG AF405 (bottom panel).

[0265] FIG. 53 provides a Western blot showing expression following transfection of expi293 cells with the indicated gB-ferritin antigen. Cells transfected with RNA encoding gB. G3-P. FuriFer NoFurin or a mock transfected control were lysed after 24 h, run on a 4-20% reducing and denaturing SDS-PAGE gel, and detected by 3 A3.

[0266] FIG. 54 provides a graph showing expression of a prefusion gB antigen design, with or without a H609Q point mutation and with three codon usages, as measured by sandwich ELISA. Expi293 cells were transfected with mRNA encoding each construct at the concentrations indicated for 24 h, then lysed. Lysate was serially diluted in and analyzed via sandwich ELISA using the indicated mouse Fc capture and human Fc detection antibodies. Data are plotted as interpolated IC50 values generated for each point in the dilution series.

[0267] FIG. 55 provides a graph showing B cell neutralization in the 4E3 cell line by sera collected on day 21 or day 35. Mice immunized on day 0 and day 21 with mRNA encoding the indicated gB antigens in addition to gp220 and gHgL-EPM-MPMV. HIV-GT2A-gp42-RtoK. Each dot indicates the neutralizing potency of sera from a single mouse.

[0268] FIG. 56 provides a graph showing epithelial cell neutralization in the HEK293T cell line by sera collected on day 21 or day 35. Mice immunized on day 0 and day 21 with mRNA encoding the indicated gB antigens in addition to gp220 and gHgL-EPM-MPMV. HIV-GT2A-gp42-RtoK. Each dot indicates the neutralizing potency of sera from a single mouse.

[0269] FIG. 57 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp220 / gp350) conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to DI 23.

[0270] FIG. 57B provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0271] FIG. 57C provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0272] FIG. 57D provides a graph showing sera binding to gp42 globular head domain conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to gp42.

[0273] FIG. 57E provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to gB.

[0274] FIG. 57F provides a graph showing sera binding to gB. D2C3 (prefusion stabilized gB) conjugated beads as quantified via Luminex. Sera was collected on day 35 from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered asmRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to gB. D2C3.

[0275] FIG. 58 provides a graph showing B cell neutralization in the 4E3 cell line by sera collected on day 21 or day 35. Mice were immunized on day 0 and day 21 with mRNA encoding the indicated antigens. Each dot indicates the neutralizing potency of sera from a single mouse.

[0276] FIG. 59 provides a graph showing epithelial cell neutralization in the HEK293T cell line by sera collected on day 21 or day 35. Mice were immunized on day 0 and day 21 with mRNA encoding the indicated antigens. Each dot indicates the neutralizing potency of sera from a single mouse.

[0277] FIG. 60 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp220 / gp350, labeled as gp220) conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to DI 23.

[0278] FIG. 60B provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0279] FIG. 60C provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0280] FIG. 60D provides a graph showing sera binding to gp42 globular head domain conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to gp42.

[0281] FIG. 60E provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered asmRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to gB.

[0282] FIG. 60F provides a graph showing sera binding to gB. G3 conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg / antigen). Each dot indicates the antibody binding of sera from a single mouse to gB. G3.

[0283] FIG. 61 provides a graph showing B cell neutralization in the 4E3 cell line by sera collected on day 21 or day 35. Rabbits were immunized on day 0 and day 21 with 7.5 pg / antigen of mRNA encoding the indicated antigens. Each dot indicates the neutralizing potency of sera from a single rabbit.

[0284] FIG. 62 provides a graph showing epithelial cell neutralization in the HEK293T cell line by sera collected on day 21 or day 35. Rabbits were immunized on day 0 and day 21 with 7.5 pg / antigen of mRNA encoding the indicated antigens. Each dot indicates the neutralizing potency of sera from a single rabbit. The dotted line at the top indicated the ULOQ, some animals had neutralizing potency too high to be accurately quantified.

[0285] FIG. 63 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp350, labeled as gp220) conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (7.5 pg / antigen). Each dot indicates the antibody binding of sera from a single rabbit to gp350.

[0286] FIG. 63B provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (7.5 pg / antigen). Each dot indicates the antibody binding of sera from a single rabbit to gH / gL / gp42.

[0287] FIG. 63C provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (7.5 pg / antigen). Each dot indicates the antibody binding of sera from a single rabbit to gH / gL.

[0288] FIG. 63D provides a graph showing sera binding to gp42 globular head domain conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigenformulations delivered as mRNA (7.5 pg / antigen). Each dot indicates the antibody binding of sera from a single rabbit to gp42.

[0289] FIG. 63E provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (7.5 pg / antigen). Each dot indicates the antibody binding of sera from a single rabbit to gB.

[0290] FIG. 63F provides a graph showing sera binding to gB. G3 conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (7.5 pg / antigen). Each dot indicates the antibody binding of sera from a single rabbit to gB. G3.

[0291] FIG. 64 provides a graph showing B cell neutralization in the 4E3 cell line by sera collected on day 21 or day 35. Guinea pigs were immunized on day 0 and day 21 with 4 pg / antigen of mRNA encoding the indicated antigens. Each dot indicates the neutralizing potency of sera from a single guinea pig.

[0292] FIG. 65 provides a graph showing epithelial cell neutralization in the HEK293T cell line by sera collected on day 21 or day 35. Guinea pigs were immunized on day 0 and day 21 with 4 pg / antigen of mRNA encoding the indicated antigens. Each dot indicates the neutralizing potency of sera from a single guinea pig. The dotted line at the top indicated the ULOQ, some animals had neutralizing potency too high to be accurately quantified.

[0293] FIG. 66 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp220 / gp350, labeled as gp220) conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from guinea pigs immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (4 pg / antigen). Each dot indicates the antibody binding of sera from a single guinea pig to D123.

[0294] FIG. 66B provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from Guinea pig immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (4 pg / antigen). Each dot indicates the antibody binding of sera from a single guinea pig to gH / gL / gp42.

[0295] FIG. 66C provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from guinea pigs immunized on days 0 and 21 with the indicated multiantigen formulationsdelivered as mRNA (4 pg / antigen). Each dot indicates the antibody binding of sera from a single guinea pig to gH / gL.

[0296] FIG. 66D provides a graph showing sera binding to gp42 globular head domain conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from guinea pigs immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (4 pg / antigen). Each dot indicates the antibody binding of sera from a single guinea pig to gp42.

[0297] FIG. 66E provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from guinea pigs immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (4 pg / antigen). Each dot indicates the antibody binding of sera from a single guinea pig to gB.

[0298] FIG. 66F provides a graph showing sera binding to gB. G3 conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from guinea pigs immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (4 pg / antigen). Each dot indicates the antibody binding of sera from a single guinea pig to gB. G3.

[0299] FIG. 67 shows sera B cell neutralization analyzed from nonhuman primates (NHPs) immunized with the indicated vaccines in the 4E3 cell line. NHPs were immunized with the indicated formulations at days 0 and 57 (indicated on the graph with grey arrows), and neutralizing potency of sera were assayed at days 0, 29, 57, 71, 85, 120, 180, 240, 298, 360, and 420. Each dot indicates the neutralizing potency of sera from a single NHP. Arrows indicate day of vaccination. Vaccinations were conducted at 25 pg per antigen in mRNA formulations or 50 pg per antigen of recombinant protein formulation.

[0300] FIG. 68 shows sera epithelial cell neutralization analyzed from nonhuman primates (NHPs) immunized with mRNA encoding the indicated antigens in the HEK-293T cell line. NHPs were immunized with the indicated formulations at days 0 and 57 (indicated on the graph with grey arrows), and neutralizing potency of sera were assayed at days 0, 29, 57, 71, 85, 120, 180, 240, 298, 360, and 420. Each dot indicates the neutralizing potency of sera from a single NHP. Arrows indicate day of vaccination. Vaccinations were conducted at 25 pg per antigen in mRNA formulations or 50 pg per antigen of recombinant protein formulation.

[0301] FIG. 69A shows sera binding to gp350 (labeled as gp220) conjugated beads, quantified via Luminex at days 0, 29, 57, 71, 85, 120, 180, and 240 following immunizationon days 0 and 57 with the indicated vaccines. Each dot indicates the antibody binding of sera from a single nonhuman primate to gp350.

[0302] FIG. 69B shows sera binding to gHgLgp42 (single chain) conjugated beads, quantified via Luminex at days 0, 29, 57, 71, 85, 120, 180, and 240 following immunization on days 0 and 57 with the indicated vaccines. Each dot indicates the antibody binding of sera from a single nonhuman primate to gH / gL / gp42.

[0303] FIG. 69C shows sera binding to gH + gL conjugated beads, quantified via Luminex at days 0, 29, 57, 71, 85, 120, 180, and 240 following immunization on days 0 and 57 with the indicated vaccines. Each dot indicates the antibody binding of sera from a single nonhuman primate to gH / gL.

[0304] FIG. 69D shows sera binding to gp42 globular head domain conjugated beads, quantified via Luminex at days 0, 29, 57, 71, 85, 120, 180, and 240 following immunization on days 0 and 57 with the indicated vaccines. Each dot indicates the antibody binding of sera from a single nonhuman primate to gp42.

[0305] FIG. 70A shows detection of antigen specific T cells, analyzed in the peripheral blood of nonhuman primates (NHPs) immunized with mRNA encoding the indicated antigens. NHPs were immunized with the indicated formulations at days 0 and 57 with 25 pg of each antigen, and PBMCs were collected at days 0, 29, 71, and 180. PBMCs were restimulated with either gp220 or gHgLgp42 single chain protein at 10 mg / mL for 24 h and analyzed using IFN-y ELISpot. Spots were counted using an ELISpot reader, and background subtraction was performed per sample by subtracting the median spot forming unit (SFU) value of medium-stimulated wells to each median SFU value of the protein-stimulated sample.

[0306] FIG. 70B shows detection of antigen specific T cells, analyzed in the peripheral blood of nonhuman primates (NHPs) immunized with mRNA encoding the indicated antigens. NHPs were immunized with the indicated formulations at days 0 and 57 with 25 pg of each antigen, and PBMCs collected at day 71 were evaluated for vaccine induced response.PBMCs were restimulated with either gp220 or gHgLgp42 single chain protein at 10 mg / mL for 24 h and analyzed for intracellular IFN-y and IL-2 expression via flow cytometry. The percentage of cytokine-producing cells was evaluated for each PBMC sample relative to an unstimulated control, and the mock-subtracted percentage of CD4 cells secreting IFN-y and / or IL-2 is plotted.

[0307] FIG. 70C shows detection of antigen specific T cells, analyzed in the peripheral blood of nonhuman primates (NHPs) immunized with mRNA encoding the indicated antigens. NHPs were immunized with the indicated formulations at days 0 and 57 with 25 pg of eachantigen, and PBMCs collected at day 71 were evaluated for vaccine indiced response.PBMCs were restimulated with either gp220 or gHgLgp42 single chain protein at 10 mg / mL for 24 h and analyzed for intracellular IFN-y and IL-2 expression via flow cytometry. The percentage of cytokine-producing cells was evaluated for each PBMC sample relative to an unstimulated control, and the mock-subtracted percentage of CD8 cells secreting IFN-y and / or IL-2 is plotted.

[0308] FIG. 71 provides a graph showing expression of gp220 mRNA encoded with three optimized codon usages and an unoptimized reference codon usage as measured by flow cytometry (top) and sandwich ELISA (bottom). Expi293 cells were transfected with mRNA encoding each construct at the concentrations indicated for 24 h. For flow cytometry, cells were harvested, stained with HB5 human Fc primary antibody followed goat anti-human IgG PE-conjugated secondary antibody, then analyzed via flow cytometry. Data are plotted as the geometric mean fluorescence intensity (gMFI) of antibody positive cells determined relative to a mock PBS transfection control. For ELISA, cells were lysed using 1% Triton X-100 in Casein blocking buffer for 4 h. Lysate was serially diluted in Casein blocking buffer and analyzed via sandwich ELISA using the indicated mouse Fc capture and human Fc detection antibodies. Data are plotted as interpolated IC50 values based on signal generated across the dilution series.

[0309] FIG. 72 provides a graph showing expression of gHgL-EPM-MPMV. HIV-GT2A-gp42-RtoK mRNA encoded with three optimized codon usages and an unoptimized reference codon usage as measured by flow cytometry (top) and sandwich ELISA (bottom). Expi293 cells were transfected with mRNA encoding each construct at the concentrations indicated for 24 h. For flow cytometry, cells were harvested, stained with CL40 human Fc primary antibody followed goat anti-human IgG PE-conjugated secondary antibody, then analyzed via flow cytometry. Data are plotted as the geometric mean fluorescence intensity (gMFI) of antibody positive cells determined relative to a mock PBS transfection control. For ELISA, cells were lysed using 1% Triton X-100 in Casein blocking buffer for 4 h. Lysate was serially diluted in Casein blocking buffer and analyzed via sandwich ELISA using the indicated mouse Fc capture and human Fc detection antibodies. Data are plotted as interpolated IC50 values based on signal generated across the dilution series.

[0310] FIG. 73 provides a graph showing expression of gB. G3-foldon-gp220tm-EPM-EABR mRNA encoded with three optimized codon usages and an unoptimized reference codon usage as measured by flow cytometry (top) and sandwich ELISA (bottom). Expi293 cells were transfected with mRNA encoding each construct at the concentrations indicated for 24h. For flow cytometry, cells were harvested, stained with AMM03 human Fc primary antibody followed goat anti-human IgG PE-conjugated secondary antibody, then analyzed via flow cytometry. Data are plotted as the geometric mean fluorescence intensity (gMFI) of antibody positive cells determined relative to a mock PBS transfection control. For ELISA, cells were lysed using 1% Triton X-100 in Casein blocking buffer for 4 h. Lysate was serially diluted in Casein blocking buffer and analyzed via sandwich ELISA using the indicated mouse Fc capture and human Fc detection antibodies. Data are plotted as interpolated IC50 values based on signal generated across the dilution series.

[0311] FIG. 74 provides a heat map showing antigenicity of gp220 / gp350 antigen designs against different monoclonal antibodies as measured by flow cytometry. Expi293 cells were transfected with mRNA encoding each construct at 1000 ng / mL (or, in the case of mock, treated with PBS) for 24 h. Cells were then harvested, stained with the indicated human Fc primary antibody followed goat anti-human IgG PE-conjugated secondary antibody, then analyzed via flow cytometry. Data are reported as percentage of antibody positive cells determined relative to the mock transfection control, with the number in each box indicating the percentage value for each antigen-antibody treatment pair.

[0312] FIG. 75 provides a heat map showing antigenicity of gH / gL / gp42 antigen designs against different monoclonal antibodies as measured by flow cytometry. Expi293 cells were transfected with mRNA encoding each construct at 1000 ng / mL (or, in the case of mock, treated with PBS) for 24 h. Cells were then harvested, stained with the indicated human Fc primary antibody followed goat anti-human IgG PE-conjugated secondary antibody, then analyzed via flow cytometry. Data are reported as percentage of antibody positive cells determined relative to the mock transfection control, with the number in each box indicating the percentage value for each antigen-antibody treatment pair.

[0313] FIG. 76 provides a heat map showing antigenicity of gB antigen designs against different monoclonal antibodies as measured by flow cytometry. Expi293 cells were transfected with mRNA encoding each construct at 1000 ng / mL or treated with PBS (mock) for 24 h. Cells were then harvested, stained with the indicated human Fc primary antibody followed goat anti-human IgG PE-conjugated secondary antibody, and analyzed via flow cytometry. Data are reported as percentage of antibody positive cells determined relative to the mock transfection control, with the number in each box indicating the percentage value for each antigen-antibody treatment pair.

[0314] FIG. 77 provides a graph showing B cell neutralization in the 4E3 cell line by sera collected on day 21 or day 35 (in black). Mice were immunized on day 0 and day 21 with 6pg of mRNA encoding the indicated antigens at the indicated ratios by mass of each mRNA component. Each dot indicates the neutralizing potency of sera from a single mouse.

[0315] FIG. 78 provides a graph showing epithelial cell neutralization in the HEK293T cell line by sera collected on day 21 or day 35 (in black). Mice were immunized on day 0 and day 21 with 6 pg of mRNA encoding the indicated antigens at the indicated ratios by mass of each mRNA component. Each dot indicates the neutralizing potency of sera from a single mouse.

[0316] FIG. 79 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp220 / gp350) conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 35 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 pg total, administered at the ratios by mass of each antigen indicated on the graph). Each dot indicates the antibody binding of sera from a single mouse to gp350.

[0317] FIG. 79B provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 35 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 pg total, administered at the ratios by mass of each antigen indicated on the graph). Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0318] FIG. 79C provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 35 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 pg total, administered at the ratios by mass of each antigen indicated on the graph). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0319] FIG. 79D provides a graph showing sera binding to gp42 globular head domain conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 35 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 pg total, administered at the ratios by mass of each antigen indicated on the graph). Each dot indicates the antibody binding of sera from a single mouse to gp42.

[0320] FIG. 79E provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 35 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered asmRNA (6 pg total, administered at the ratios by mass of each antigen indicated on the graph). Each dot indicates the antibody binding of sera from a single mouse to gB.

[0321] FIG. 79F provides a graph showing sera binding to gB. G3 conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 35 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 pg total, administered at the ratios by mass of each antigen indicated on the graph). Each dot indicates the antibody binding of sera from a single mouse to gB. G3.

[0322] FIG. 79G provides a graph showing sera binding to gB. D2C3 (prefusion stabilized gB) conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 35 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 pg total, administered at the ratios by mass of each antigen indicated on the graph). Each dot indicates the antibody binding of sera from a single mouse to gB. D2C3.

[0323] FIG. 80 provides a graph showing B cell neutralization in the 4E3 cell line by sera collected on day 21 or day 35. Mice were immunized on day 0 and day 21 with the indicated doses of mRNA (per antigen) encoding the indicated constructs. Each dot indicates the neutralizing potency of sera from a single mouse.

[0324] FIG. 81 provides a graph showing epithelial cell neutralization in the HEK293T cell line by sera collected on day 21 or day 35. Mice were immunized on day 0 and day 21 with the indicated doses of mRNA (per antigen) encoding the indicated constructs. Each dot indicates the neutralizing potency of sera from a single mouse. The upper dotted line denotes the ULOQ, as in some cases animals were too potent of neutralizers to determine an IC50, so the dots are positioned at the LTLOQ.

[0325] FIG. 82 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp220 / gp350) conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg or 0.4 pg / antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, coformulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single mouse to DI 23.

[0326] FIG. 82B provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg or 0.4 pg / antigen, administered either as mRNAsformulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0327] FIG. 82C provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg or 0.4 pg / antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0328] FIG. 82D provides a graph showing sera binding to gp42 globular head domain conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg or 0.4 pg / antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single mouse to gp42.

[0329] FIG. 82E provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg or 0.4 pg / antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single mouse to post-fusion gB.

[0330] FIG. 82F provides a graph showing sera binding to gB. G3 conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (2 pg or 0.4 pg / antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single mouse to gB. G3.

[0331] FIG. 83 provides a graph showing B cell neutralization in the 4E3 cell line by sera collected on day 21 or day 35. Rabbits were immunized on day 0 and day 21 with 7.5 pg mRNA (per antigen) encoding the indicated constructs. IWhere indicated, RNAs encoding the antigens listed were coformulated in a single LNP. Each dot indicates the neutralizing potency of sera from a single rabbit.

[0332] FIG. 84 provides a graph showing epithelial cell neutralization in the HEK293T cell line by sera collected on day 21 or day 35. Rabbits were immunized on day 0 and day 21with 7.5 pg mRNA (per antigen) encoding the indicated constructs. Where indicated, RNAs encoding the antigens listed were coformulated in a single LNP. Each dot indicates the neutralizing potency of sera from a single rabbit. The upper dotted line denotes the ULOQ, as in some cases animals were too potent of neutralizers to determine an IC50, so the dots are positioned at the ULOQ.

[0333] FIG. 85 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp220 / gp350) conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (7.5 pg per antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1: 1: 1 mass ratio). Each dot indicates the antibody binding of sera from a single rabbit to DI 23.

[0334] FIG. 85B provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (7.5 pg per antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single rabbit to gH / gL / gp42.

[0335] FIG. 85C provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (7.5 pg per antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single rabbit to gH / gL.

[0336] FIG. 85D provides a graph showing sera binding to gp42 globular head domain conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (7.5 pg per antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single rabbit to gp42.

[0337] FIG. 85E provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered asmRNA (7.5 µg per antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single rabbit to post-fusion gB.

[0338] FIG. 85F provides a graph showing sera binding to gB. G3 conjugated beads as quantified via Luminex. Sera was collected on day 21 (top panel) or day 35 (bottom panel) from rabbits immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (7.5 pg per antigen, administered either as mRNAs formulated as separate LNPs or, where indicated, co-formulated into a single LNP at a 1:1:1 mass ratio). Each dot indicates the antibody binding of sera from a single rabbit to gB. G3.

[0339] FIG. 86 provides a graph showing (left) cell surface and (right) secreted expression of various gHgLgp42 antigens containing the indicated EPM motifs. Expi293 cells were transfected with 1 mg / mL of RNAs encoding the indicated antigens for 24 h. Supernatants were collected to evaluate secreted antigen expression, and cells were subsequently stained with 3E8 human Fc primary antibody and goat anti-human IgG PE secondary antibody to evaluate cell surface expression. Cell surface expression was measured by flow cytometry and plotted as median fluorescence intensity (MFI). Meanwhile, supernatants were diluted with Casein Blocking buffer and analyzed using a cytometric bead assay with bead-conjugated AMM01 mouse Fc as the capture antibody and 3E8 human Fc as the detection antibody. Goat anti-human IgG PE was used as a secondary antibody. Secreted antigen captured by AMM01 beads and detected by 3E8-PE was measured by flow cytometry and plotted as median fluorescence intensity (MFI).

[0340] FIG. 87 provides a graph showing (left) cell surface and (right) secreted expression of various gB. D2C3 antigens containing the indicated EPM motifs. Expi293 cells were transfected with 1 mg / mL of RNAs encoding the indicated antigens for 24 h. Supernatants were collected to evaluate secreted antigen expression, and cells were subsequently stained with AMM05 human Fc primary antibody and goat anti-human IgG PE secondary antibody to evaluate cell surface expression. Cell surface expression was measured by flow cytometry and plotted as median fluorescence intensity (MFI). Meanwhile, supernatants were diluted with Casein Blocking buffer and analyzed using a cytometric bead assay with bead-conjugated AMM05 mouse Fc as the capture antibody and 3A5 human Fc as the detection antibody. Goat anti-human IgG PE was used as a secondary antibody. Secreted antigen captured by AMM05 beads and detected by 3 A5-PE was measured by flow cytometry and plotted as median fluorescence intensity (MFI).

[0341] FIG. 88 provides a graph showing B cell neutralization in the 4E3 cell line by sera collected on day 28. Mice were immunized on day 0 and day 21 with the indicated doses of mRNA (6 pg or 1.5 pg total) encoding the indicated constructs and formulated at a 1:1:1 mass ratio. Each dot indicates the neutralizing potency of sera from a single mouse.

[0342] FIG. 89 provides a graph showing epithelial cell neutralization in the HEK293T cell line by sera collected on day 21 or day 28. Mice were immunized on day 0 and day 21 with the indicated doses of mRNA (6 pg or 1.5 pg total) encoding the indicated constructs and formulated at a 1:1:1 mass ratio. Each dot indicates the neutralizing potency of sera from a single mouse. The upper dotted line denotes the ULOQ, as in some cases animals were too potent of neutralizers to determine an IC50, so the dots are positioned at the ULOQ.

[0343] FIG. 90 A provides a graph showing sera binding to DI 23 (the receptor binding domain of gp220 / gp350) conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 28 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 or 1.5 pg total, administered at a 1:1:1 mass ratio of the indicated constructs). Each dot indicates the antibody binding of sera from a single mouse to DI 23.

[0344] FIG. 90B provides a graph showing sera binding to gHgLgp42 (single chain) conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 28 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 or 1.5 pg total, administered at a 1:1:1 mass ratio of the indicated constructs). Each dot indicates the antibody binding of sera from a single mouse to gH / gL / gp42.

[0345] FIG. 90C provides a graph showing sera binding to gH + gL conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 28 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 or 1.5 pg total, administered at a 1: 1: 1 mass ratio of the indicated constructs). Each dot indicates the antibody binding of sera from a single mouse to gH / gL.

[0346] FIG. 90D provides a graph showing sera binding to gp42 globular head domain conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 28 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 or 1.5 pg total, administered at a 1:1:1 mass ratio of the indicated constructs). Each dot indicates the antibody binding of sera from a single mouse to

[0347] FIG. 90E provides a graph showing sera binding to gB conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 28 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 or 1.5 pg total, administered at a 1:1:1 mass ratio of the indicated constructs). Each dot indicates the antibody binding of sera from a single mouse to gB.

[0348] FIG. 90F provides a graph showing sera binding to gB. D2C3 (prefusion stabilized gB) conjugated beads as quantified via Luminex. Sera was collected on day 21 (bottom panel) or day 28 (top panel) from mice immunized on days 0 and 21 with the indicated multiantigen formulations delivered as mRNA (6 or 1.5 pg total, administered at a 1:1:1 mass ratio of the indicated constructs). Each dot indicates the antibody binding of sera from a single mouse to gB. D2C3.

[0349] FIG. 91 provides a table showing an in-silico analysis of predicted immunogenicity of EPM motifs derived from mouse and common degu.DETAILED DESCRIPTION

[0350] Disclosed herein are engineered Epstein Barr Virus (EBV) polypeptides, modified EBV glycoprotein B (gB), modified EBV glycoprotein H (gH), modified EBV glycoprotein L (gL), modified EBV glycoprotein 42 (gp42), modified EBV glycoprotein 350 (gp350), modified EBV glycoprotein BMRF-2 (BMRF-2), polynucleotides encoding said polypeptides and glycoproteins, related vaccine compositions, and related methods for making and using the compositions described herein.Epstein Barr Virus

[0351] Epstein Barr Virus (EBV) can infect both B cells and epithelial cells and has numerous proteins on its surface that are responsible for viral engagement or fusion with these target cells. For viral cell engagement, gp350 (or a spliced form of this protein, gp220) is responsible for binding to CD21 or CD35 on B cells, gH / gL / gp42 is a heterotrimer responsible for binding to HLA-II (through gp42) on B cells and EphA2, R9AP, DSC-2, and DSC-3 (through gH / gL) on epithelial cells, and BMRF-2 is a multipass transmembrane glycoprotein that binds a3, a5,aV, and pi integrins on epithelial cells. Upon B and / or epithelial cell engagement, an additional glycoprotein, gB, undergoes a conformational change which facilitates viral fusion to these target cells. Given the role of these EBVglycoproteins in viral engagement and fusion with host cells, as shown in FIG. 1A, they are antigens of interest for vaccine design.Engineered EBV polypeptides

[0352] In one aspect, disclosed herein are engineered EBV polypeptides comprising at least one modified EBV glycoprotein. The modified EBV glycoprotein comprises at least one modification relative to a wild type EBV glycoprotein and may function as an EBV antigen. In some embodiments, the at least one modified EBV glycoprotein comprises a modified EBV gB described herein, a modified EBV gH described herein, a modified EBV gL described herein, a modified EBV gp42 described herein, a modified EBV gH / gL described herein, a modified EBV gH / gL / gp42 described herein, a modified EBV gp350 described herein, a modified EBV BMRF-2 described herein, or a combination thereof.

[0353] In certain embodiments, the at least one modified EBV glycoprotein comprises a modified EBV gB described herein. In certain embodiments, the at least one modified EBV glycoprotein comprises a modified EBV gH described herein. In certain embodiments, the at least one modified EBV glycoprotein comprises a modified EBV gL described herein. In certain embodiments, the at least one modified EBV glycoprotein comprises a modified EBV gH described herein and a modified EBV gL described herein. In certain embodiments, the at least one modified EBV glycoprotein comprises a modified EBV gH / gL described herein. In certain embodiments, the at least one modified EBV glycoprotein comprises a modified EBV gp42 described herein. In certain embodiments, the at least one modified EBV glycoprotein comprises a modified EBV gH described herein, a modified EBV gL described herein, and a modified gp42 described herein. In certain embodiments, the at least one modified EBV glycoprotein comprises a modified EBV gp350 described herein. In certain embodiments, the at least one modified EBV glycoprotein comprises a modified EBV BMRF-2 described herein.

[0354] In certain embodiments, the engineered EBV polypeptides of the disclosure comprise a modified EBV gB described herein. In certain embodiments, the engineered EBV polypeptides of the disclosure comprise a modified EBV gH described herein. In certain embodiments, the engineered EBV polypeptides of the disclosure comprise a modified EBV gL described herein. In certain embodiments, the engineered EBV polypeptides of the disclosure comprise a modified EBV gp42 described herein. In certain embodiments, the engineered EBV polypeptides of the disclosure comprise a modified EBV gp350 describedherein. In certain embodiments, the engineered EBV polypeptides of the disclosure comprise a modified EBV BMRF-2 described herein.

[0355] In some embodiments, the at least one modification in the modified EBV glycoprotein relative to a wild type EBV glycoprotein comprises a deletion, an insertion, or an amino acid substitution.Insertion

[0356] In some embodiments, the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion. In some embodiments, the insertion comprises insertion of a ribosomal skip site, an endocytosis prevention motif (EPM), a signal sequence, an endoplasmic reticulum (ER) export sequence, an ESCRT recruiting domain (ERD), an ESCRT-independent eVLP inducing domain, a transmembrane (TM) domain, a dimerization domain, a trimerization domain, a ferritin sequence, an EBV polypeptide or portion thereof, a linker, a cleavage site, a furin cleavage site, an EBV T-cell epitope, a signal peptide, or a combination thereof.Endosomal sorting complex required for transport (ESCRT) recruiting domain (ERD)

[0357] Enveloped virus-like particles are referred to herein as eVLPs and are discussed in further detail below. The eVLPs of the present disclosure are non-infectious membraned particles whose production does not require a viral capsid protein and is instead driven by one or more polypeptides that induces eVLP formation and release from the cell when expressed. A polypeptide that induces eVLP formation may comprise an ESCRT recruiting domain (ERD) or an ESCRT-independent eVLP inducing domain. In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of an ESCRT recruiting domain (ERD).

[0358] ERDs are dependent on host ESCRT machinery. In some embodiments, the ERD is selected from: (A) viral ERDs, (B) non -human homolog ERDs, (C) synthetic ERDs comprising a backbone capable of forming a coiled-coil structure, also referred to as synthetic coiled-coil ERDs, (D) synthetic ERDs comprising at least two ESCRT -recruiting motifs (ERMs), also referred to as synthetic ERDs with ERMs, and (E) tandem ERDs. Examples of polypeptides that depend on host ESCRT machinery to drive eVLPs formation include, but are not limited to, those disclosed in Int. Pub. No. WO2022261230A1 and Int. App. No.PCT / US2025 / 031513, the contents of which are incorporated herein in their entirety by reference.

[0359] In some embodiments, the ERD comprises an ESCRT- and ALIX-binding region (EABR) sequence, which facilitates interactions with TSG101 and ALIX, two proteins that are involved in the recruitment of ESCRT machinery to sites of eVLP budding. An EABR sequence recruits host ESCRT proteins to the modified EBV glycoprotein at the plasma membrane and results in the self-assembly and budding of eVLPs bearing the modified EBV glycoprotein described herein. Table 2D provides amino acid sequences for exemplary ERDs that can be incorporated into the engineered EBV polypeptides of the present disclosure. In some embodiments, the ERD comprises a sequence in Table 2D or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ERD comprises the amino acid sequence of any one of SEQ ID NOS:70-108 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.ESCRT-independent eVLP inducing domain

[0360] In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of an ESCRT-independent eVLP inducing domain. An ESCRT-independent eVLP inducing domain comprises a polypeptide that promotes the selfassembly and budding of eVLPs bearing the modified EBV glycoprotein described herein through a mechanism that is entirely independent of host ESCRT machinery. ESCRT-independent eVLP inducing domains are described, for example, in Int. App. No.PCT / US2025 / 039970, the contents of which are incorporated herein in their entirety by reference. Non-limiting examples of ESCRT-independent eVLP inducing domains include amphipathic helices, tetraspanins, and the platelet-derived growth factor receptor transmembrane domain.

[0361] In some embodiments, the ESCRT-independent eVLP inducing domain is derived from (1) an envelope protein of a virus, or a domain or fragment thereof; (2) a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof; (3) a lysosome-associated membrane protein 2 (LAMP2) polypeptide, or a domain or fragment thereof, or a binding partner thereof; (4) a polypeptide that binds to phosphatidylserine, or a domain or fragment thereof; (5) a platelet-derived growth factor receptor (PDGFR) polypeptide, or adomain or fragment thereof; and / or (6) a polypeptide capable of forming an amphipathic helix. In some embodiments, the ESCRT-independent eVLP inducing domain comprises an amphipathic helix or the platelet-derived growth factor receptor transmembrane domain.Endocytosis prevention motif (EPM)

[0362] Without being held to theory, eVLP production may be enhanced by preventing endocytosis of the engineered EBV polypeptide or the modified EBV glycoproteins described herein and extend the duration for which it remains at the plasma membrane. In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of an endocytosis prevention motif (EPM). Antigens which are retained for a longer duration at the plasma membrane may provide more opportunities for interaction with ESCRT proteins and subsequent eVLP formation. Non-limiting examples of species from which an Fc gamma receptor protein EPM sequence may be derived include house mouse (Mus miisciilus. common degu (Octodon degus), alpine marmot (Marmota mar motet), or golden spiny mouse (Acomys russatus). In some embodiments, the EPM sequence derived from a host species may be truncated or mutated to improve activity, reduce unwanted activity, or remove human identity or homology.

[0363] In some embodiments, the EPM is derived from a Fc gamma receptor II (FcgR-II). In some embodiments, the EPM derived from a FcgR-II comprises a modification. In some embodiments, the modification is an amino acid deletion. In certain embodiments, the amino acid deletion comprises a truncation.

[0364] In certain embodiments, the EPM is derived from Mouse FcgR-II. In certain embodiments, the EPM is derived from common degu FcgR-II. In certain embodiments, the EPM is derived from alpine marmot FcgR-II. In certain embodiments, the EPM is derived from golden spiny mouse FcgR-II. In some embodiments, the EPM comprises a modification corresponding to a modification set forth in SEQ ID NO: 62 relative to SEQ ID NO: 61. In some embodiments, the EPM comprises a modification corresponding to a modification set forth in SEQ ID NO: 66 relative to SEQ ID NO: 60.

[0365] In some embodiments, the EPM comprises an amino acid sequence listed in Table 2C or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the EPM comprises the amino acid sequence of any one of SEQ ID NOS: 60-66 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Ribosomal skip site

[0366] In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of a ribosomal skip site. In some embodiments, the ribosomal skip site comprises a 2A peptide, also referred to as a 2A site. The 2A site can either allow for ribosomal read through, generating a full-length polypeptide, or cause the ribosome to pause during translation, generating two shorter polypeptides. In exemplary embodiments, the 2A peptide is derived from foot-and-mouth disease virus, equine rhinitis virus, porcine teschovirus-1, or Thosea asigna virus. The 2 A peptide derived from foot-and-mouth disease virus is referred to as F2A. The 2A peptide derived from equine rhinitis A virus is referred to as E2A. The 2A peptide derived from porcine teschovirus-1 is referred to as P2A. The 2A peptide derived from Thosea asigna virus is referred to as T2A. In some embodiments, a GSG linker sequence is added to the 2A peptide. The linker may enhance the cleavage efficiency at the 2 A site. Exemplary 2 A peptide sequences are provided in Table 2B below. In some embodiments, the 2A peptide comprises a sequence in Table 2B or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the 2A peptide comprises the amino acid sequence of any one of SEQ ID NOS: 50-57 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. Without being held to theory, using 2A peptides may allow for increased control of the ratio at which the two shorter polypeptides are expressed during a ribosomal skip event.Signal peptide (SP)

[0367] In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of a signal peptide. In some embodiments, the signal peptide is a peptide segment of about 10 to 40 amino acids. When fused to an engineered EBV polypeptide of the present disclosure, the signal peptide is capable of targeting the polypeptide to an organelle, to the plasma membrane, or for secretion from a cell. In some embodiments, the signal peptide targets the engineered EBV polypeptide to the endoplasmicreticulum (ER). In certain embodiments, the signal peptide is the signal peptide from human IgE. In certain embodiments, the signal peptide is the signal peptide from IGVH. In certain embodiments, the signal peptide is the signal peptide from human tissue plasminogen activator (tPA). In certain embodiments, the signal peptide is an isoform of the signal peptide from human CD5. In certain embodiments, the signal peptide is the signal peptide from IGKV. In certain embodiments, the signal peptide is the signal peptide from human albumin.

[0368] Exemplary signal peptide sequences are provided in Table 2A below. In some embodiments, the signal peptide comprises an amino acid sequence in Table 2A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOS: 40-47 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Transmembrane (TM) domains

[0369] In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of a transmembrane (TM) domain. Insertion of a transmembrane domain may anchor an otherwise secreted protein to the plasma membrane and / or anchor portions of a protein to the membrane. In some cases, the TM domain can stabilize the protein into a desired conformation. Insertion of TM domains are discussed in further detail below. An insertion of a TM domain into an engineered EBV polypeptide or a modified EBV glycoprotein described herein results in an engineered EBV polypeptide or a modified EBV glycoprotein comprising a TM domain that is also referred to herein as a heterologous TM domain.

[0370] In some embodiments, the TM domain is derived from EBV gp220. In some embodiments, the TM domain is derived from Influenza neuraminidase (NA). In some embodiments wherein the TM domain is derived from influenza NA, the signal peptide is removed or replaced. In some embodiments, the TM domain is a two-pass TM domain. In some embodiments, the two-pass TM domain is derived from the Zika virus NS2 protein. In some embodiments, the two-pass TM domain is derived from the influenza virus NA protein. TM domain derived from Zika virus NS2 protein. In some embodiments, the TM domain is derived from Measles virus hemagglutinin.

[0371] Exemplary TM domain sequences are provided in Table 2E. In some embodiments, the TM domain comprises an amino acid sequence in Table 2E or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the TM domain comprises the amino acid sequence of any one of SEQ ID NOS: 120-125, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Trimerization domains

[0372] In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of a trimerization domain. Insertion of a trimerization domain may stabilize the trimeric structure of a modified EBV glycoprotein, thereby improving its antigenicity or immunogenicity. Insertion of trimerization domains are discussed in further detail below. Exemplary trimerization domain sequences are provided in Table 2F. In some embodiments, the trimerization domain comprises an amino acid sequence in Table 2F or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the trimerization domain comprises the amino acid sequence of any one of SEQ ID NOS: 170-174, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Ferritins

[0373] In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of a ferritin sequence. The ferritin sequence may be inserted at the C terminal or the N terminal of the modified EBV glycoprotein. In some embodiments, the ferritin sequence is inserted at the C terminal of the modified EBV glycoprotein. In exemplary embodiments, the ferritin sequence is inserted at the C terminal of the modified EBV glycoprotein after another modification to the glycoprotein, such as a truncation. In some embodiments, the ferritin comprises a ferritin derived from an amphibian, a bacterium, a fungus, an insect, or a plant. In some embodiments, the ferritin comprises a ferritin derived from a bacterium. Non-limiting examples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H.cetorum, H. vulpis, C. coli, E. coli, andB. subtilis. In some embodiments, the ferritin is derived from H. pylori, U urealycitum, E. coli, M. tuberculosis, P. furiosus, C. tepidum, or V. cholera. In some embodiments, the ferritin is derived from E. coli, H. pylori, or P. furiosus. In some embodiments, the ferritin is derived from E. coli. In some embodiments, the ferritin is derived from H. pylori. In some embodiments, the ferritin is derived from P. furiosus. In some embodiments, the ferritin is derived from bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog ferritin. A hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from a bacterium. For example, a hybrid bullfrog ferritin may comprise a bullfrog ferritin and a ferritin derived from E. coli, H. pylori, or P. furiosus, referred to as a hybrid bullfrog- / ' / Coli ferritin, a hybrid bullfrog- / / , pylori ferritin, or a hybrid bullfrog- / / furiosus ferritin, respectively. In some embodiments, the ferritin comprises a hybrid bullfrog- / / . Coli ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / , pylori ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / / furiosus ferritin. Variations can be made in the amino acid sequence of a ferritin without affecting its ability to self-assemble into an oligomer or a nanoparticle. Such variations include insertion of amino acid residues, deletions of amino acid residues, or substitutions of amino acid residues. For example, the sequence of a monomeric ferritin subunit can be derived from a mammalian ferritin amino acid sequence but be divergent enough from the naturally occurring sequence such that when administered to a mammalian subject of the species from which the mammalian ferritin amino acid sequence was derived, it does not result in the production of antibodies that react with the natural ferritin protein of the mammal. Exemplary ferritin sequences are provided in Table 4A. In some embodiments, the ferritin comprises an amino acid sequence in Table 4A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Cleavage sites

[0374] In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of a cleavage site. In exemplary embodiments, the cleavage site is a furin cleavage site, also referred to as “furin site” herein. The furin cleavage site is a motif recognized by the protease furin. In general, the furin cleavage site occurs after a basic amino acid. In some embodiments, the furin cleavage occurs after a basic acid amino, such as arginine (R) or Lysine (K). In some embodiments, the furin cleavage site motif is R-X-K / R-R. Exemplary sequences for furin cleavage sites are provided in Table 2H. In some embodiments, the furin cleavage site comprises an amino acid sequence in Table 2H or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the furin cleavage site comprises the amino acid sequence of any one of SEQ ID NOS: 190-195 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.EBV T cell epitopes

[0375] In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of a EBV T cell epitope. A EBV T cell epitope is a part of an EBV protein, such as the EBV glycoproteins described herein, that can be recognized by T cells. The EBV T cell epitope may be a predicted or known epitope. Predictions can be done utilizing standard T cell epitope predictors. For example, a list of T cell epitope predictor tools is available at the Immune Epitope Database (IEDB) Analysis Resource, including various tools for immunogenicity prediction, MHC binding, processing, and other structure tools. Alternatively, known epitopes that have been identified from EBV infected individuals could be used (see, for example, Hislop et al., J Exp Med. 2002;195(7):893-905, Chen et al., J Virol. 2021;95(10):e00081-21, Duraiswamy et al., J Virol. 2003;77(13):7401-10, Catalina et al., J Immunol. 2001;167(8):4450-7, Rist et al., J Virol. 2015;89(l):703-12, and Drosu et al., PNAS 2024;121(44):e2416097121). T cell epitopes can be immunodominant or non-immunodominant. They can be targeted by CD8+ T cells or targeted by CD4+ T cells, with restriction by any human leukocyte antigen (HLA) alleles that are present in the human population.Linkers

[0376] In some embodiments where the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid insertion, the insertion comprises insertion of a linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is a Gly-Ser linker. In some embodiments, the linker is derived from EBV gp350 or BMRF2. In some embodiments, the linker is flanked by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 glycines on its N terminal, C terminal, or both. Exemplarylinker sequences are provided in Table 2G. In some embodiments, the linker comprises an amino acid sequence in Table 2G or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOS: 180-188 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Deletion

[0377] In some embodiments, the modified EBV glycoprotein comprises a deletion. In some embodiments, the deletion comprises deletion of at least one amino acid residue at the C-terminal, N-terminal, and / or a middle portion of the protein relative to the wild type glycoprotein. In some embodiments, the deletion comprises deletion of at least 2 consecutive amino acid residues or at least 2 non-consecutive amino acid residues. In some embodiments, the deletion comprises deletion of at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 consecutive amino acids. In some embodiments, the deletion comprises a truncation, where at least one amino acid is deleted from the N-terminal or C-terminal of the glycoprotein.

[0378] In some embodiments, the deletion comprises deletion of at least one amino acid within a furin cleavage site in the EBV glycoprotein. In some embodiments, the deletion eliminates the furin cleavage site. In some embodiments, the deletion comprises deletion of a cleavage site. In exemplary embodiments, the cleavage site is a furin cleavage site.Exemplary sequences for furin cleavage sites are provided in Table 2H. In some embodiments, the deletion comprises deletion of a furin cleavage site comprising an amino acid sequence in Table 2H or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the deletion comprises deletion of a furin cleavage site comprising the amino acid sequence of any one of SEQ ID NOS: 190-195 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Substitution

[0379] In some embodiments, the at least one modification in the modified EBV glycoprotein relative to the wild type EBV glycoprotein comprises an amino acid substitution. In someembodiments, the substitution comprises substitution of at least one amino acid residue at the C-terminal, N-terminal, and / or a middle portion of the protein relative to the wild type glycoprotein. In some embodiments, the substitution comprises substitution of at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 amino acids.

[0380] In some embodiments, the amino acid substitution comprises substitution of at least one amino acid within a furin cleavage site in the EBV glycoprotein. In some embodiments, the amino acid substitution within the furin cleavage site eliminates said cleavage site. A furin cleavage may occur after a basic acid amino, such as arginine (R) or Lysine (K) within a furin cleavage site. In some embodiments, the substitution comprises substitution of at least one amino acid within a furin cleavage site comprising the motif R-X-K / R-R. Exemplary sequences for furin cleavage sites are provided in Table 2H. In some embodiments, the substitution comprises substitution of at least one amino acid within a furin cleavage site comprising an amino acid sequence in Table 2H or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the substitution comprises substitution of at least one amino acid within a furin cleavage site comprising the amino acid sequence in any one of SEQ ID NOS: 190-195 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Improved characteristics

[0381] The engineered EBV polypeptides of the present disclosure may exhibit at least one improved characteristic relative to a wild type EBV polypeptide. The at least one improved characteristic may comprise (a) increased stability, (b) increased antigenicity, (c) increased immunogenicity, (d) increased secretion, (e) increased secretion as enveloped virus-like particles (eVLPs), (f) increased secretion as ferritin nanoparticles, (g) increased retention on the cell surface, (h) increased cell-surface expression, (i) increased exposure of target epitopes, (j) decreased exposure of off-target epitopes, (k) decreased off target immune response, (1) increased number of neutralizing epitopes targeted, (m) ability to express multiple EBV glycoproteins from a single polypeptide, (n) increased control of the rate of expression of the modified EBV glycoproteins, (o) removal of a disease-associated epitope, (p) increased neutralization potency, (q) increased neutralization potency relative to totalimmunogenicity, (r) increased number of epitopes targeted, (s) improved thermal stability, (t) increased stabilization in a prefusion, prefusion-like, or postfusion conformation or a combination thereof. In some embodiments, the at least one improved characteristic comprises removal of a disease-associated epitope. In certain embodiments, the disease-associated epitope is a semi-heterologous disease associated epitope. These improved characteristics are described in further detail below and demonstrated in the Examples.Modified EBV glycoproteinsgB

[0382] In another aspect, disclosed herein is a modified EBV glycoprotein B (gB), wherein the modified EBV gB comprises an EBV gB amino acid sequence comprising at least one modification relative to a wild type EBV gB; and optionally wherein the modified EBV gB exhibits an improved characteristic relative to a wild type EBV gB. Improved characteristics are described in further detail herein. In some embodiments, the improved characteristic is increased stabilization in a prefusion, prefusion-like, or post-fusion conformation. In some embodiments, the improved characteristic is increased stability, increased antigenicity, increased immunogenicity, or a combination thereof. In some embodiments, the modified EBV gB is derived from the B-95.8 strain or the M81 strain of EBV. In certain embodiments, the modified EBV gB is derived from the B-95.8 strain of EBV. The full-length sequence for wild type EBV gB protein from the B-95.8 strain comprises the sequence set forth in SEQ ID NO: 20. In certain embodiments, the modified EBV gB is derived from the M81 strain of EBV. The full-length sequence for wild type EBV gB protein from the M81 strain comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the at least one modification relative to a wild type EBV gB comprises an amino acid insertion, an amino acid deletion, an amino acid substitution, or a combination thereof. In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid insertion, an amino acid deletion, an amino acid substitution, or a combination thereof, relative to a wild type EBV gB.

[0383] EBV gB has five domains referred to as domain I (D-I), domain II (D-II), domain III (D-III), domain IV (D-IV), and domain V (D-V). The sequences of the five gB domains of the B-95.8 strain are provided in Table 1C. In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid sequence in Table 1C or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the modified EBV gBs of the disclosure comprise the amino acid sequence of any one of SEQ IDNOS: 20-28 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Conformation

[0384] EBV gB is natively a trimer on the surface of the Epstein Barr virus. It exists initially in the prefusion conformation, but upon engagement with a target cell (B cell or epithelial cell), fusion of the viral membrane and host membrane is triggered and gB rearranges into the post-fusion conformation. When gB is expressed in the absence of a virus, the gB protein is often unstable and rapidly rearranges into the post-fusion conformation.

[0385] In some embodiments, the modified EBV gBs of the disclosure have a post-fusion like conformation. Without being held to theory, it is thought that immune response against the post-fusion conformation may not be as productive because the gB protein is presented in the form it would naturally occur in after membrane fusion rather than the form required for membrane fusion on an infectious viral particle. In some embodiments, the modified EBV gBs of the disclosure have a prefusion conformation. It should be understood that prefusion molecules, or molecules in the prefusion form, exist in a thermodynamically unstable state that promotes the fusion event of the virus to the host cell, resulting in the transition from the prefusion state to the post-fusion state. During the transition from prefusion to post-fusion state, a protein may transition through a number of intermediate forms.

[0386] In some embodiments, the modified EBV gBs of the disclosure have a prefusion-like conformation. A “prefusion-like” conformation, as used herein, refers to a conformation which comprises at least one structural feature of a prefusion gB protein and / or refers to a state in which gB exists prior to viral membrane fusion with the target membrane about 10-100% of the time, including intermediate forms. A prefusion-like conformation also encompasses a conformation of an EBV gB which has been engineered to increase its stability in a state in which gB exists prior to viral membrane fusion (also referred to herein as a “prefusion stabilized” conformation).

[0387] Strategies for stabilizing EBV gB in a prefusion or prefusion-like conformation may include using inter-protomeric disulfide bonds, flexible loop stabilization, and cavity-filling substitutions. EBV gB in prefusion and prefusion-like forms stabilized using these approaches are described, for example, in McCool et al, bioRXiv. 2025.05.19.654955 (“McCool et al”), McCallum et al. bioRxiv. 2024.10.23.619923 (“McCallum et al”), and International App. No. PCT / US2023 / 069411, all of which are incorporated herein byreference in their entirety. A modified EBV gB of the disclosure may comprise a prefusion or a prefusion-like EBV gB described in McCool et al, such as Variant G3 and Variant D2C3. In some embodiments, the modified EBV gBs of the disclosure comprise Variants G3, also referred to as gB. G3 in the present disclosure, or Variant D2C3, also referred to as gB. D2C3 in the present disclosure, as described in McCool et al.

[0388] In some embodiments of the modified gBs of the disclosure, the at least one modification relative to a wild type EBV gB comprises an amino acid substitution. In some embodiments, the modified EBV gBs of the disclosure comprises an amino acid substitution relative to a wildtype EBV gB.

[0389] In some embodiments, the modified gBs of the disclosure comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid substitutions at positions corresponding to positions 402-452 relative to the wildtype EBV gB sequence set forth in SEQ ID NO: 21. In some embodiments, the modified gBs of the disclosure comprises a sequence comprising amino acid substitutions at positions corresponding to positions 402-452 relative to SEQ ID NO: 21. In certain embodiments, the amino acids at positions corresponding to positions 402-452 relative to SEQ ID NO: 21 are substituted with a linker sequence. In exemplary embodiments, the amino acids at positions corresponding to positions 402-452 relative to SEQ ID NO: 21 are substituted with the sequence GSPPGSPP.

[0390] In some embodiments, the modified gBs of the disclosure comprises an amino acid substitution at a position corresponding to position A293 relative to SEQ ID NO: 21. In certain embodiments, the amino acid substitution at position A293 is A293P.

[0391] In some embodiments, the modified gBs of the disclosure comprises an amino acid substitution at positions corresponding to positions Q527 and E634 relative to SEQ ID NO: 21. In certain embodiments, the amino acid substitutions at positions Q527 and E634 are Q527C and E634C, respectively. Without being held to theory or mechanism, mutations corresponding to Q527C and E634C may lead to formation of a disulfide bond between the two cysteines.

[0392] In some embodiments, the modified gBs of the disclosure comprises a sequence comprising one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid substitutions at positions corresponding to positions 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196,D220, R428, R429, R430, R431, R432, H316, D320, S325, Q527, E634, A293, orH609 relative to SEQ ID NO: 21.

[0393] In some embodiments, the modified gBs of the disclosure comprises a sequence comprising one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid substitutions at positions corresponding to positions 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, R432, H316, D320, S325, or H609 relative to SEQ ID NO: 21.

[0394] In some embodiments, the modified gBs of the disclosure comprises a sequence comprising an amino acid substitution at a position corresponding to position H609 relative to SEQ ID NO: 21. In certain embodiments, the amino acid substitution at position H609 is H609Q.

[0395] In some embodiments, the modified gBs of the disclosure comprises a sequence comprising an amino acid substitution at positions corresponding to positions 189, L628, A175, and E634 relative to SEQ ID NO: 21. In certain embodiments, the amino acid substitutions at positions 189, L628, A175, and E634 are I89C, L628GCG, A175C, and E634C, respectively.

[0396] In some embodiments, the modified gBs of the disclosure comprises a sequence comprising an amino acid substitution at positions corresponding to positions 189, L628, A175, E634, H316, D320, and S325 relative to SEQ ID NO: 21. In certain embodiments, the amino acid substitutions at positions 189, L628, A175, E634, H316, D320, and S325 are I89C, L628GCG, A175C, E634C, H316I, D320Q, and S325L, respectively.

[0397] In some embodiments, the modified gBs of the disclosure comprises a sequence comprising one or more amino acid substitutions, relative to SEQ ID NO: 21, selected from the group consisting of: I89C, L628GCG, A175C, E634C, W112H, Y113R, W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, R432G, H316I, D320Q, S325L, and H609Q.

[0398] In exemplary embodiments, the modified gBs of the disclosure comprises a sequence comprising amino acid substitutions W112H, Y113R, W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, and R432G, relative to SEQ ID NO: 21.

[0399] In exemplary embodiments, the modified gBs of the disclosure comprises a sequence comprising amino acid substitutions I89C, L628GCG, A175C, E634C, W112H, Y113R,W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, and R432G relative to SEQ ID NO: 21.

[0400] In exemplary embodiments, the modified gBs of the disclosure comprises a sequence comprising amino acid substitutions I89C, L628GCG, A175C, E634C, W112H, Y113R, W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, R432G, H316I, D320Q, and S325L relative to SEQ ID NO: 21.

[0401] In exemplary embodiments, the modified gBs of the disclosure comprises a sequence with amino acid substitutions I89C, L628GCG, A175C, E634C, W112H, Y113R, W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, R432G, H316I, D320Q, S325L, and H609Q relative to SEQ ID NO: 21.

[0402] In some embodiments, the modified gBs of the disclosure comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 28, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0403] In some embodiments, the modified gBs of the disclosure comprises amino acid substitutions at positions corresponding to at least two of S54C, S55C, H56C, G172C, A175C, V178C, G227C, G322C, S325C, D478C, A480C, A515C, V529C, D564C, L580C, Y644C, L696C, and S727C relative to SEQ ID NO: 20. In some embodiments, the amino acid substitutions comprise one or two or three or more pairs of amino acid substitutions selected from the group consisting of: S54C and A515C, S55C and G227C, H56C and G227C, A175C and V529C, G322C and A480C, S325C and A480C, G322C and D478C, L696C and S727C, A175C and V178C, G172C and D564C, and L580C and Y644C, relative to SEQ ID NO: 20. In some embodiments, native cysteine amino acid residues are substituted with a non-Cysteine amino acid residue, such as Ala, Arg, As, Asp, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai.

[0404] Fusion of the EB V viral membrane and host membrane requires a dramatic move of the gB fusion loop from the viral membrane in order to engage the host membrane.Therefore, another approach to stabilizing gB in a prefusion like conformation is the insertion of at least one TM domain into a gB fusion loop. Without being held to theory or mechanism, in some embodiments, insertion of a TM domain stabilizes gB in a prefusion or prefusionlike conformation.

[0405] In some embodiments of the modified EBV gBs of the disclosure, the at least one modification relative a wild type EBV gB comprises an amino acid insertion. In some embodiments of the modified EBV gBs of the disclosure, the at least one modificationrelative a wild type EBV gB comprises an amino acid insertion of a transmembrane (TM) domain. In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid insertion of a TM domain in a fusion loop of EBV gB. In some embodiments, the at least one modification relative a wild type EBV gB comprises an amino acid substitution of a fusion loop of EBV gB or a portion thereof with a transmembrane (TM) domain. Exemplary sequences of EBV gB fusion loops which can be substituted with a TM domain or into which a TM domain may be inserted include GW YA (SEQ ID NO: 175) and WLIWT (SEQ ID NO: 176).

[0406] In some embodiments, the modified EBV gBs of the disclosure comprise an insertion of a TM domain into one or more sequences corresponding to the anchor site sequence(s) underlined in the wild type gB sequence of the B-95.8 EBV strain below, with * denoting a translation stop codon:

[0407] MTRRRVLSVVVLLAALACRLGAQ [anchor site 3JTPEQPAPPATTVQPTAT RQQTSFPFRVCELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSY TKIVTNILIYNG_[anchor site 1JWYADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKD GLTRVYVDRD [anchor site 4] GVNITVNLKPTGGLANGVRRYASQTELYDAPG WLI[anchor site 2JWTYRTRTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKN KETFHERADSFHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCP LQHWQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKTMHEK YEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELTTPTSSPPSSPSPP APSAARGSTPAAVLRRRRRDAGNATTPVPPTAPGKSLGTLNNPATVQIQFAYDSLRR QINRMLGDLARAWCLEQKRQNMVLRELTKINPTTVMSSIYGKAVAAKRLGDVISVS QCVPVNQATVTLRKSMRV PG[anchor site 5J S ETMCYSRPLVSFSFIND [anchor site 6JTKTYEGQLGTDNE IFLTKKMTEVCQATSQYYFQSGNEIHVYNDYHHFKTIELDGIATLQTFISLNTSLIENI DFASLELYSRDEQRASNVFDLEGIFREYNFQAQNIAGLRKDLDNAVSNGRNQFVDGL GELMDSLGSVGQSITNLVSTVGGLFSSLVSGFISFFKNPFGGMLILVLVAGVVILVISL TRRTRQMSQQPVQMLYPGIDELAQQHASGEGPGINPISKTELQAIMLALHEQNQEQK RAAQRAAGPSVASRALQAARDRFPGLRRRRYHDPETAAALLGEAETEF* (SEQ ID NO: 20).

[0408] The TM domain may be inserted before or after any one of the amino acids within the underlined anchor sites indicated above. The TM domain may also be inserted in between any two amino acids within the fusion loop or any two amino acids within 10 amino acids ofone of the six anchor sites indicated in the wild type gB sequence above or a corresponding anchor site in a modified EBV gB glycoprotein described herein. Anchor sites 1-6 indicated above in brackets provide exemplary positions where the TM domain may be inserted.

[0409] Anchor site 1 is within the sequence of GW YA, and anchor site 2 is within the sequence of WLIWT. The sequences GWYA and WLIWT correspond to the fusion loops of the gB protein. When the wild type gB protein above is expressed on the surface, such as on the viral membrane or an eVLP or a ferritin nanoparticle, it is expected that in the prefusion form, anchor site 3 is about 40A from the membrane. Anchor site 1 is about 55A from the membrane. Anchor sites 2, 4, and 5 are less than 10A from the membrane and anchor site 6 is about 30A from the membrane. In some embodiments, a modified gB protein which has a prefusion conformation has an anchor site 3 that is about 40A from the membrane, an anchor site 1 that is about 55A from the membrane, an anchor site 2, 4, or 5 that is less than 10A from the membrane, and / or an anchor site 6 that is about 30A from the membrane.

[0410] In addition to anchoring domains to a membrane to stabilize EBV gB in a prefusion or prefusion-like conformation, insertion of a TM domain may also be used to attach an EBV gB without a TM domain (for example, an engineered EBV gB ectodomain) to a membrane independently or in combination with domain anchoring. As discussed above, in some embodiments, the modified EBV gBs of the disclosure comprises an EBV gB having a prefusion, prefusion-like, or post-fusion conformation.

[0411] An EBV gB engineered for stabilization in a prefusion, prefusion-like, or post-fusion conformation may comprise an EBV gB ectodomain which does not contain a TM domain. For example, EBV gB Variants G3 and D2C3 described in McCool et al are engineered gB ectodomains which do not contain a TM domain. For cell surface expression of a protein without a TM domain, a heterologous TM domain may be inserted into said protein to enable anchoring of the protein to a cell membrane. Insertion of a heterologous domain may support the incorporation of an ERD and / or an EPM, which in some embodiments is necessary for generation of eVLPs displaying on its surface said protein or a portion thereof. Accordingly, in some embodiments, the modified EBV gBs of the disclosure comprise an insertion of a TM domain at the C-terminus of an EBV gB ectodomain having a prefusion, prefusion-like, or post-fusion conformation. In certain embodiments, the EBV gB ectodomain comprises the sequence or SEQ ID NO: 27 or SEQ ID NO: 28 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the EBV gB ectodomain comprises a sequence corresponding to amino acids 1-688 of SEQ ID NO: 21 ora sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the TM domain is inserted at the C-terminus of a wild-type, a prefusion, a prefusion-like, or a post-fusion form of an EBV gB ectodomain.

[0412] In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid substitution of the native membrane proximal external region (MPER), TM domain, and / or the C-terminal domain of a wild type EBV gB with a heterologous TM domain.

[0413] In some embodiments of the modified gBs of the disclosure, the at least one modification relative to a wild type EBV gB comprises an amino acid substitution of the MPER, TM domain, and / or the C-terminal domain corresponding to that of EBV gB from the B-95.8 EBV strain or the M81 EBV strain with a heterologous TM domain.

[0414] In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid substitution wherein:a) a native TM domain corresponding to that of a wildtype gB is substituted with a heterologous TM domain; orb) a native TM domain and a native C-terminal domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain.

[0415] In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid substitution wherein a native TM domain corresponding to that of a wildtype gB is substituted with a heterologous TM domain. In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid substitution wherein a native TM domain and a native C-terminal domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain.

[0416] In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid substitution wherein:a) a native membrane proximal external region (MPER) and a native TM domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain; or b) a native membrane proximal external region (MPER), a native TM domain, and a native C-terminal domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain.

[0417] In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid substitution wherein a native membrane proximal external region (MPER) and a native TM domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain. In some embodiments, the modified EBV gBs of the disclosure comprise an aminoacid substitution wherein a native membrane proximal external region (MPER), a native TM domain, and a native C-terminal domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain.

[0418] In both the B-95.8 EBV strain or the M81 EBV strain, the MPER and TM domain correspond to amino acids 685-754 relative to SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the TM domain used for insertion or substitution into a modified gB of the disclosure (i.e., a heterologous TM domain) is derived from a viral protein or a bacterial protein. In some embodiments, the modified gBs of the disclosure comprise an insertion of a TM domain and a trimerization motif. In some embodiments, the modified gBs of the disclosure comprise an insertion of a TM domain and do not comprise a trimerization motif.

[0419] In some embodiments, the TM domain used for insertion or substitution into a modified gB of the disclosure (i.e., a heterologous TM domain) is derived from EBV gp220. In some embodiments, the TM domain is derived from Influenza neuraminidase (NA). In some embodiments wherein the TM domain is derived from influenza NA, the signal peptide is removed or replaced. In some embodiments, the TM domain is a two-pass TM domain. In some embodiments, the two-pass TM domain is derived from the Zika virus NS2 protein. In some embodiments, the two-pass TM domain is derived from the influenza virus NA protein. In some embodiments, the TM domain is derived from Measles virus hemagglutinin. In some embodiments, the TM domain comprises a sequence in Table 2E or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the TM domain comprises the amino acid sequence of any one of SEQ ID NOS: 120-125, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0420] The TM domain may also be flanked by a linker on its C terminus, N terminus, or both the C and the N terminal ends. In some embodiments where the modified EBV gBs of the disclosure comprise insertion of a TM domain, the modified gB further comprises an insertion of a linker at the C terminus, N terminus, or at the C and N terminals of the TM domain. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a peptide linker. In exemplary embodiments, the peptide linker is a Gly-Ser linker. In some embodiments, the length of the linker corresponds approximately to the distance between the insertion site and the membrane. When a protein (for example, EBV gB) changes from the prefusion to post-fusion conformation, the distance between an insertion site and the membrane changes. This change is most likely an increase in distance. Without being held totheory, selection of an appropriate linker length, by proxy, can prevent the protein from transitioning from a prefusion to a post-fusion conformation, for example, if the linker is not long enough to accommodate the post-fusion form. In some embodiments, the length of the linker is between 1 and 40 amino acids. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 amino acids in length. In some embodiments, the linker is more than 40 amino acids in length.Other modifications

[0421] In some embodiments of the modified EBV gBs of the disclosure, the at least one modification in the modified EBV gB comprises an insertion, a deletion, a substitution, or a combination thereof. In some embodiments, the at least one modification comprises an insertion. In some embodiments, the at least one modification comprises an insertion of an ERD, an ESCRT-independent eVLP inducing domain, an EPM, a signal peptide, a trimerization domain, a peptide tag, a TM domain, a ferritin sequence, or a combination thereof. In some embodiments, the insertion comprises an ERD. In some embodiments, the insertion comprises an EPM. In some embodiments, the insertion comprises insertion of an ERD and an EPM. In some embodiments, the insertion comprises insertion of:

[0422] a. a TM domain;

[0423] b. an ERD and a TM domain;

[0424] c. an ERD, a TM domain, and a trimerization domain;

[0425] d. an ERD, a signal peptide, a TM domain, and a trimerization domain;

[0426] e. an ERD, an EPM, and a signal peptide;

[0427] f. an ERD, an EPM, a signal peptide, and a TM domain;

[0428] g. an ERD, an EPM, a TM domain, and a trimerization domain;

[0429] h. an ERD, an EPM, a signal peptide, a TM domain, and a trimerization domain;

[0430] i. an ESCRT-independent eVLP inducing domain and a TM domain;

[0431] j. an ESCRT-independent eVLP inducing domain, a signal peptide, and a TM domain;

[0432] k. an ESCRT-independent eVLP inducing domain, an EPM, and a signal peptide; or

[0433] 1. an ESCRT-independent eVLP inducing domain, an EPM, a signal peptide, and a TM domain.

[0434] The different domains and components of (a) through (1) may be inserted in any order.

[0435] In exemplary embodiments, the signal peptide is inserted at the N-terminus of the modified EBV gB.

[0436] In exemplary embodiments, the EPM is N-terminal to the ERD or ESCRT-independent eVLP inducing domain. In exemplary embodiments, the ERD or ESCRT-independent eVLP inducing domain is C-terminal to the EPM.

[0437] In exemplary embodiments, the TM domain is N-terminal to the ERD or ESCRT-independent eVLP inducing domain. In exemplary embodiments, the ERD or ESCRT-independent eVLP inducing domain is C-terminal to the TM domain.

[0438] In exemplary embodiments, the TM domain is N-terminal to the EPM. In exemplary embodiments, the EPM is C-terminal to the TM domain.

[0439] In exemplary embodiments, the trimerization domain is N-terminal to the TM domain. In exemplary embodiments, the TM domain is C-terminal to the trimerization domain.

[0440] ERDs are discussed in further detail herein and any ERD described herein may be inserted into a modified EBV gB of the disclosure. In some embodiments, the ERD comprises a sequence in Table 2D or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ERD comprises the amino acid sequence of any one of SEQ ID NOS:70-108 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0441] EPMs are discussed in further detail herein and any EPM described herein may be inserted into a modified EBV gB of the disclosure. In some embodiments, the EPM is derived from a Fc gamma receptor II (FcgR-II). In certain embodiments, the EPM is derived from mouse, common degu, alpine marmot, or golden spiny mouse FcgR-II. In some embodiments, the EPM comprises an amino acid sequence in Table 2C or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the EPM comprises the amino acid sequence of any one of SEQ ID NOS: 60-66 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0442] In some embodiments, the insertion comprises an ESCRT-independent eVLP inducing domain. Non-limiting examples of ESCRT-independent eVLP inducing domains include amphipathic helices, tetraspanins, and the platelet-derived growth factor receptor transmembrane domain. In some embodiments, the ESCRT-independent eVLP inducing domain comprises a sequence in Table 2J or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% sequence identity thereto. In some embodiments, the ESCRT-independent eVLP inducing domain comprises the amino acid sequence of any one of SEQ ID NOS: 500-504 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0443] In some embodiments, the insertion comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence in Table 2A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOS: 40-47 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0444] In some embodiments, the insertion comprises a trimerization domain. In some embodiments, the trimerization domain is derived from the dimeric GCN4 protein of yeast. In some embodiments, the trimerization domain is derived from the foldon trimerization domain found in fibritin protein of T4 bacteriophage. In some embodiments, the trimerization domain comprises a sequence in Table 2F or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the trimerization domain comprises the amino acid sequence of any one of SEQ ID NOS: 170-173, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. Without being held to theory or mechanism, insertion of a trimerization domain may stabilize the trimeric structure of a modified EBV gB, thereby improving its antigenicity or immunogenicity. In some embodiments of the modified EBV gBs of the disclosure, the at least one modification comprises insertion of a trimerization domain and the improved characteristic is increased antigenicity and / or increased immunogenicity.Ferritin

[0445] In some embodiments, the insertion comprises insertion of a ferritin. Ferritins are discussed in further detail herein and any ferritin described herein may be inserted into a modified EBV gB of the disclosure. The ferritin may be inserted at the C terminal or the N terminal of a modified EBV gB of the disclosure. In some embodiments, the ferritin comprises a ferritin derived from an amphibian, a bacterium, a fungus, or a plant. In some embodiments, the ferritin comprises a ferritin derived from a bacterium. Non-limitingexamples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H. cetorum, H. vulpis, C. coli, E. coli, andB. subtilis. In some embodiments, the ferritin is derived from E. coli, H. pylori, or P. furiosus. In some embodiments, the ferritin is derived from E. coli. In some embodiments, the ferritin is derived from H. pylori. In some embodiments, the ferritin is derived from P. furiosus. In some embodiments, the ferritin is derived from bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / :. Coli ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / / , pylori ferritin. Exemplary ferritin sequences are provided in Table 4A. In some embodiments, the ferritin comprises an amino acid sequence in Table 4A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ferritin comprises the amino acid sequence of any one of SEQ ID NOS: 440-452 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Removal of a disease-associated epitope

[0446] In some embodiments of the modified EBV gBs of the disclosure, the improved characteristic is removal of an epitope associated with a disease. In some embodiments, the epitope is a T cell epitope associated with increased risk of a disease. In certain embodiments, the disease is multiple sclerosis (MS). In certain embodiments, the epitope comprises a peptide motif.

[0447] In some embodiments of the modified EBV gBs of the disclosure, the at least one modification relative to a wild type EBV gB comprises an amino acid deletion or amino acid substitution of a histidine residue within the GNEIHVYNDYHHFKT peptide motif. The GNEIHVYNDYHHFKT peptide motif corresponds to the amino acids at positions 599-613 relative to the sequence of SEQ ID NO: 21. In exemplary embodiments, the at least one modification relative to a wild type EBV gB comprises an amino acid deletion or amino acid substitution of a histidine residue within the GNEIHVYNDYHHFKT peptide motif and the improved characteristic is removal of a disease-associated epitope. In certain embodiments, the disease-associated epitope is associated with MS.

[0448] In some embodiments of the modified EBV gBs of the disclosure, the at least one modification relative to a wild type EBV gB comprises an amino acid deletion. In someembodiments, the amino acid deletion comprises a deletion of a histidine residue within a GNEIHVYNDYHHFKT peptide motif. In certain embodiments, the deletion comprises deletion of histidine (H) at position 609, relative to SEQ ID NO: 21. In certain embodiments, an engineered EBV polypeptide described herein comprising a deletion of a histidine residue within the GNEIHVYNDYHHFKT peptide motif exhibits an improved characteristic, wherein the improved characteristic is removal of a disease-associated epitope. In exemplary embodiments, the disease-associated epitope is associated with MS.

[0449] In some embodiments, the at least one modification relative to a wild type EBV gB comprises an amino acid substitution. In some embodiments, the at least one modification comprises an amino acid substitution of a histidine residue within the GNEIHVYNDYHHFKT peptide motif. In certain embodiments, the at least one modification comprises an amino acid substitution of a histidine residue within the GNEIHVYNDYHHFKT peptide motif with a glutamine residue. In exemplary embodiments, the substitution comprises substitution of the histidine at position 609, relative to SEQ ID NO: 21, with another amino acid. In exemplary embodiments, the substitution comprises substitution of histidine (H) at position 609 (referred to as H609), relative to SEQ ID NO: 21, for a glutamine (Q). Substitution of the histidine at position 609, relative to SEQ ID NO: 21, for a glutamine is referred to as H609Q herein, as present in SEQ ID NO: 340.

[0450] It can be appreciated that the substitution of amino acids, such as the histidine at position 609 relative to SEQ ID NO: 21, is a modification that may be applied to any gB sequence comprising said amino acids at positions corresponding to those relative to SEQ ID NO: 21. For example, the substitution of the histidine at position 609 relative to SEQ ID NO: 21 is a modification that may be applied to any gB comprising a histidine at the position corresponding to position 609 relative to SEQ ID NO:21. A gB sequence may be a prefusion gB, prefusion-like gB, or post-fusion gB sequence. In some embodiments, the modified EBV gBs of the disclosure comprise a prefusion gB sequence comprising a H609 amino acid substitution. In certain embodiments, the modified EBV gBs of the disclosure comprise a prefusion gB sequence comprising a H609Q amino acid substitution. In some embodiments, the modified EBV gBs of the disclosure comprise a prefusion-like gB sequence comprising a H609 amino acid substitution. In certain embodiments, the modified EBV gBs of the disclosure comprise a prefusion-like gB sequence comprising a H609Q amino acid substitution. In some embodiments, the modified EBV gBs of the disclosure comprise a postfusion gB sequence comprising aH609 amino acid substitution. In certain embodiments, the modified EBV gBs of the disclosure comprise a H609Q amino acid substitution.

[0451] In some embodiments, the position of an amino acid that is deleted or substituted may not be at the same position relative to SEQ ID NO: 21 due to other modifications made to a gB sequence, but the position of the amino acid corresponding to that relative to SEQ ID NO: 21 can be identified through methods such as sequence alignment. As an example, the position of the histidine at position 609 relative to SEQ ID NO: 21 that is deleted or substituted may not be at position 609 due to other modifications, but the position of the histidine corresponding to H609 can be identified through sequence alignment. In some embodiments, the modified EBV gBs of the disclosure comprise a prefusion gB sequence, wherein the prefusion gB sequence comprises an amino substitution corresponding to the H609 amino acid substitution. In certain embodiments, the prefusion gB sequence comprises an amino acid substitution corresponding to the H609Q substitution. In some embodiments, the modified EBV gBs of the disclosure comprise a prefusion-like gB sequence, wherein the prefusion-like gB sequence comprises an amino substitution corresponding to the H609 amino acid substitution. In certain embodiments, the prefusion-like gB sequence comprises an amino acid substitution corresponding to the H609Q substitution. In some embodiments, the modified EBV gBs of the disclosure comprise a post-fusion gB sequence, wherein the post-fusion gB sequence comprises an amino substitution corresponding to the H609 amino acid substitution. In certain embodiments, the post-fusion gB sequence comprises an amino acid substitution corresponding to the H609Q substitution.

[0452] In some embodiments of the modified EBV gBs of the disclosure, the at least one modification comprises a deletion of at least one amino acid residue at the C-terminal, N-terminal, and / or middle portions of the protein compared to the wild type gB protein. In some embodiments, the deletion comprises deletion of at least one of the five gB domains (D-I, D-II, D-III, D-IV, and D-V). In some embodiments, the deletion comprises deletion of 2, 3, or 4 of the 5 gB domains.

[0453] In some embodiments, the deletion comprises deletion of at least one amino acid within a furin cleavage site in the gB protein. In some embodiments, the deletion eliminates the cleavage site (renders the cleavage site nonfunctional). In some embodiments, the deletion comprises deletion of a furin cleavage site comprising the motif R-X-K / R-R.Exemplary sequences for furin cleavage sites are provided in Table 2H. In some embodiments, the deletion comprises deletion of a furin cleavage site comprising an amino acid sequence in Table 2H or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the deletion comprises deletion of a furincleavage site comprising the amino acid sequence of any one of SEQ ID NOS: 190-195 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0454] In some embodiments of the modified EBV gBs of the disclosure, the at least one modification relative to a wild type EBV gB comprises an amino acid substitution. In some embodiments, the at least one modification comprises amino acid substitution of at least one of the amino acids within a fusion loop of EBV gB. In some embodiments, the at least one modification comprises amino acid substitution of at least one of the amino acids within a EBV gB fusion loop having a sequence of GWYA (SEQ ID NO: 175) or WLIWT (SEQ ID NO: 176).

[0455] In some embodiments, the modified EBV gB comprises amino acid substitution of a wildtype EBV gB fusion loop with a fusion loop of a different viral protein. In certain embodiments, the different viral protein is a protein from Herpes simplex virus (HSV)-l or HSV-2. In certain embodiments, the different viral protein is HSV-1 or HSV-2 gB. In some embodiments, the at least one modification comprises substitution of a wildtype EBV gB fusion loop or a portion thereof with a HSV-2 gB fusion loop or a portion thereof. In some embodiments, the HSV-2 gB fusion loop comprises the sequence GHRA (SEQ ID NO: 177) or RVEAT (SEQ ID NO: 178).

[0456] In some embodiments, the modified EBV gB comprises substitution of the wildtype EBV gB fusion loop sequence of GWYA with the sequence GHRA. In some embodiments, the modified EBV gB comprises substitution of the wildtype EBV gB fusion loop sequence of WLIWT with the sequence RVEAT. In some embodiments, the modified EBV gB comprises substitution of the wildtype EBV gB fusion loop sequence of GWYA with the sequence GHRA and substitution of the wildtype EBV gB fusion loop sequence of WLIWT with the sequence RVEAT. In some embodiments, the modified EBV gB comprises amino acid substitution of a wildtype EBV gB fusion loop with a linker selected from Table 2G. In some embodiments, the modified EBV gB comprises amino acid substitution of a wildtype EBV gB fusion loop with a linker comprising the sequence of any one of SEQ ID NOS: ISO-188.

[0457] In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid substitution of at least one amino acid within a furin cleavage site in the gB protein. In some embodiments, the amino acid substitution within the furin cleavage site eliminates said cleavage site (renders the cleavage site nonfunctional). A furin cleavage may occur after a basic acid amino, such as arginine (R) or Lysine (K) within a furin cleavage site. In someembodiments, the substitution comprises substitution of at least one amino acid within a furin cleavage site comprising the motif R-X-K / R-R. Exemplary sequences for furin cleavage sites are provided in Table 2H. In some embodiments, the substitution comprises substitution of at least one amino acid within a furin cleavage site comprising an amino acid sequence in Table 2H or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the substitution comprises substitution of at least one amino acid within a furin cleavage site comprising the amino acid sequence in any one of SEQ ID NOS: 190-195 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.TM domain anchors

[0458] Also provided herein is an engineered EBV gB protein comprising a heterologous transmembrane (TM) domain, wherein the heterologous TM domain is capable of anchoring the gB protein. Without being held to theory or mechanism, in some embodiments, insertion of a TM domain stabilizes gB in a prefusion or prefusion-like conformation. In some embodiments, the TM domain is inserted into a fusion loop of EBV gB, as described above. In some embodiments, the TM domain is inserted into an anchor site described above. The TM domain may be inserted in between any two amino acids within the EBV gB fusion loop or any two amino acids within 10 amino acids of one of the six anchor sites indicated in the wild type gB sequence above or a corresponding anchor site in a modified EBV gB glycoprotein described herein. In some embodiments, the TM domain comprises a sequence in Table 2E or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the TM domain comprises the amino acid sequence of any one of SEQ ID NOS: 120-125, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0459] In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid sequence in Table 1C, Table 3C, or Table 3F, or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0460] In some embodiments, the modified EBV gBs of the disclosure comprises the amino acid sequence in any one of SEQ ID NOS: 20-28, 220-224, 230-231, 270-283, and 340-391 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.gp350

[0461] In another aspect, disclosed herein is a modified EBV glycoprotein 350 (gp350), wherein the modified EBV gp350 comprises an EBV gp350 amino acid sequence comprising at least one modification relative to a wild type EBV gp350; and optionally wherein the modified EBV gp350 exhibits an improved characteristic relative to a wild type EBV gp350. The gp350 glycoprotein has a spliced form referred to as gp220. Additionally, a truncated form of gp350 composed of domain I (D-I), domain II (D-II), and domain III of gp350 is referred to herein as D123. As used herein, the term “gp350” encompasses gp350 and all truncated or spliced forms thereof, including gp220 and D123, unless otherwise indicated. The sequences of gp350 (full length), g220, DI 23 and gp350 domain I, domain II, and domain III are provided in Table ID.In some embodiments, the modified EBV gp350s of the disclosure comprise an amino acid sequence in Table ID or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the modified EBV gp350s of the disclosure comprise the amino acid sequence in any one of SEQ ID NOS: 30-36 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0462] In some embodiments, the at least one modification relative to a wild type EBV gp350 comprises an insertion, a deletion, a substitution, or a combination thereof. In some embodiments, the at least one modification comprises an amino acid insertion. In some embodiments, the insertion comprises an ERD, an ESCRT-independent eVLP inducing domain, an EPM, a signal peptide, a peptide tag, or a combination thereof. In some embodiments, the insertion comprises an ERD. In some embodiments, the insertion comprises an EPM. In some embodiments, the insertion comprises insertion of an ERD and an EPM. In some embodiments, the insertion comprises insertion of:

[0463] a. a signal peptide;

[0464] b. an ERD and a signal peptide;

[0465] c. an EPM and a signal peptide

[0466] d. an ERD, an EPM, and a signal peptide;

[0467] e an ESCRT-independent eVLP inducing domain and a signal peptide; or

[0468] f. an ESCRT-independent eVLP inducing domain, an EPM, and a signal peptide.

[0469] The different domains and components of (a) through (f) may be inserted in any order.

[0470] In exemplary embodiments, the EPM is N-terminal to the ERD or ESCRT-independent eVLP inducing domain. In exemplary embodiments, the ERD or ESCRT-independent eVLP inducing domain is C-terminal to the EPM.

[0471] In some embodiments, the insertion comprises an ERD. ERDs are discussed in further detail herein and any ERD described herein may be inserted into a modified EBV gp350 of the disclosure. In some embodiments, the ERD comprises a sequence in Table 2D or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ERD comprises the amino acid sequence of any one of SEQ ID NOS:70-108 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0472] In some embodiments, the insertion comprises an EPM. EPMs are discussed in further detail herein and any EPM described herein may be inserted into a modified EBV gp350 of the disclosure. In some embodiments, the EPM is derived from a Fc gamma receptor II (FcgR-II). In certain embodiments, the EPM is derived from mouse, common degu, alpine marmot, or golden spiny mouse FcgR-II. In some embodiments, the EPM comprises an amino acid sequence in Table 2C or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the EPM comprises the amino acid sequence of any one of SEQ ID NOS: 60-66 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0473] In some embodiments, the insertion comprises an ESCRT-independent eVLP inducing domain. ESCRT-independent eVLP inducing domains are discussed in further detail herein and any ESCRT-independent eVLP inducing domains described herein may be inserted into a modified EBV gp350 of the disclosure. Non-limiting examples of ESCRT-independent eVLP inducing domains include amphipathic helices and the platelet-derived growth factor receptor transmembrane domain. In some embodiments, the ESCRT-independent eVLP inducing domain comprises a sequence in Table 2J or a sequence havingat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ESCRT-independent eVLP inducing domain comprises the amino acid sequence of any one of SEQ ID NOS: 500-504 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0474] In some embodiments, the insertion comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence in Table 2A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOS: 40-47 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Ferritin

[0475] In some embodiments, the insertion comprises insertion of a ferritin. Ferritins are discussed in further detail herein and any ferritin described herein may be inserted into a modified EBV gp350 of the disclosure. The ferritin may be inserted at the C terminal or the N terminal of the modified EBV gp350 protein. In some embodiments, the ferritin comprises a ferritin derived from an amphibian, a bacterium, a fungus, or a plant. In some embodiments, the ferritin comprises a ferritin derived from a bacterium. Non-limiting examples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H. cetorum, H. vulpis, C. coli, E. coli, andB. subtilis. In some embodiments, the ferritin is derived from E. coli, H. pylori, or P. furiosus. In some embodiments, the ferritin is derived from E. coli. In some embodiments, the ferritin is derived from H. pylori. In some embodiments, the ferritin is derived from P. furiosus. In some embodiments, the ferritin is derived from bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / :. Coli ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / / . pylori ferritin.

[0476] Exemplary ferritin sequences are provided in Table 4A. In some embodiments, the ferritin comprises an amino acid sequence in Table 4A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ferritincomprises the amino acid sequence of any one of SEQ ID NOS: 440-452 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Deletion

[0477] In some embodiments of the modified EBV gp350s of the disclosure, the at least one modification comprises a deletion of at least one amino acid residue at the C-terminal, N-terminal, and / or middle portions of the EBV gp350 sequence relative to a wild type gp350 sequence. In some embodiments, the deletion comprises deletion of a gp350 domain, such as gp350 D-I, D-II, or D-III. In some embodiments, the deletion comprises a C-terminal truncation relative to a wild type gp350. In some embodiments, the C-terminal truncation comprises deletion of a consecutive sequence at least 25 amino acids in length from the C-terminal of a wild type gp350. In some embodiments, the C terminal truncation comprises deletion of a sequence at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 95 amino acids, at least 100 amino acids, at least 105 amino acids, at least 110 amino acids, at least 115 amino acids, at least 120 amino acids, at least 125 amino acids, at least 130 amino acids, at least 135 amino acids, at least 140 amino acids, at least 145 amino acids, or at least 150 amino acids in length from the C-terminal of a wild type gp350.

[0478] In some embodiments, the at least one modification comprises an amino acid substitution. In some embodiments, the substitution comprises substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 amino acids relative to a wild type gp350.

[0479] In some embodiments, the modified EBV gBs of the disclosure comprise an amino acid sequence in Table ID, Table 3D, or Table 3G, or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0480] In some embodiments, the modified EBV gBs of the disclosure comprises the amino acid sequence in any one of SEQ ID NOS: 30-36, 225-229, 290-293, and 400-409 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. gH, gL, gp42gH

[0481] In another aspect, disclosed herein is a modified EBV glycoprotein H (gH), wherein the modified EBV gH comprises an EBV gH amino acid sequence comprising at least one modification relative to a wild type EBV gH; and optionally wherein the modified EBV gH exhibits an improved characteristic relative to a wild type EBV gH. The gH glycoprotein has four domains referred to as domain I (D-I), domain II (D-II), domain III (D-III), and domain IV (D-IV). The sequences of a wild type EBV gH protein and the four gH domains are provided in Table 1A. In some embodiments, the at least one modification relative to a wild type EBV gH comprises an insertion, a deletion, a substitution, or a combination thereof.

[0482] In some embodiments, the at least one modification comprises an insertion. In some embodiments, the insertion comprises insertion of a ferritin. The ferritin may be inserted at the C terminal or the N terminal of the modified EBV gH protein. In some embodiments, the ferritin comprises a ferritin derived from an amphibian, a bacterium, a fungus, or a plant. In some embodiments, the ferritin comprises a ferritin derived from a bacterium. Non-limiting examples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H. cetorum, H. vulpis, C. coli, E. coli, andB. subtilis. In some embodiments, the ferritin is derived from E. coli, H. pylori, or P. furiosus. In some embodiments, the ferritin is derived from E. coli. In some embodiments, the ferritin is derived from H. pylori. In some embodiments, the ferritin is derived from P. furiosus. In some embodiments, the ferritin is derived from bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / :. Coli ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog-77, pylori ferritin. Exemplary ferritin sequences are provided in Table 4A. In some embodiments, the ferritin comprises an amino acid sequence in Table 4A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ferritin comprises the amino acid sequence of any one of SEQ ID NOS: 440-452 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0483] In some embodiments, the at least one modification comprises a deletion of at least one amino acid residue at the C-terminal, N-terminal, and / or middle portions of the EBV gH sequence relative to a wild type gH. In some embodiments, the deletion comprises deletion of a gH domain, such as gH domains D-I, D-II, D-III, or D-IV. In some embodiments, thedeletion comprises deletion of at least 25 amino acids relative to a wild type gH protein. In some embodiments, the deletion comprises deletion of at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 95 amino acids, at least 100 amino acids, at least 105 amino acids, at least 110 amino acids, at least 115 amino acids, at least 120 amino acids, at least 125 amino acids, at least 130 amino acids, at least 135 amino acids, at least 140 amino acids, at least 145 amino acids, or at least 150 amino acids relative to a wild type gH protein.

[0484] In some embodiments, the modified EBV gHs of the disclosure comprise a sequence in Table 1A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0485] In some embodiments, the modified EBV gHs of the disclosure comprise a sequence of any one of SEQ ID NOs: 1-5 and 210-211 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.gL

[0486] In another aspect, disclosed herein is a modified EBV glycoprotein L (gL), wherein the modified EBV gL comprises an EBV gL amino acid sequence comprising at least one modification relative to a wild type EBV gL; and optionally wherein the modified EBV gL exhibits an improved characteristic relative to a wild type EBV gL. In some embodiments, the at least one modification relative to a wild type EBV gL comprises an insertion, a deletion, a substitution, or a combination thereof. In some embodiments, the at least one modification comprises an insertion.

[0487] In some embodiments, the insertion comprises insertion of a ferritin. The ferritin may be inserted at the C terminal or the N terminal of the modified EBV gL protein. In some embodiments, the ferritin comprises a ferritin derived from an amphibian, a bacterium, a fungus, or a plant. In some embodiments, the ferritin comprises a ferritin derived from a bacterium. Non-limiting examples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H. cetorum, H. vulpis, C. coli, E. coli, and B. subtilis. In some embodiments, the ferritin is derived from E. coli, H. pylori, or P. furiosus. In some embodiments, the ferritin is derived from E. coli. Insome embodiments, the ferritin is derived from H. pylori. In some embodiments, the ferritin is derived from P.furiosus. In some embodiments, the ferritin is derived from bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / :. Coli ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / / , pylori ferritin. Exemplary ferritin sequences are provided in Table 4A. In some embodiments, the ferritin comprises an amino acid sequence in Table 4A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ferritin comprises the amino acid sequence of any one of SEQ ID NOS: 440-452 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0488] In some embodiments, the at least one modification comprises a deletion of at least one amino acid residue at the C-terminal, N-terminal, and / or middle portions of the EBV gL sequence relative to a wild type gL. In some embodiments, the deletion comprises deletion of at least 10 amino acids relative to a wild type gL. In some embodiments, the deletion comprises deletion of at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 95 amino acids, or at least 100 amino acids relative to a wild type gL. In some embodiments, the modified EBV gLs of the disclosure comprise the sequence of SEQ ID NO: 6 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.gp42

[0489] In another aspect, disclosed herein is a modified EBV glycoprotein 42 (gp42), wherein the modified EBV gp42 comprises an EBV gp42 amino acid sequence comprising at least one modification relative to a wild type EBV gp42; and optionally wherein the modified EBV gp42 exhibits an improved characteristic relative to a wild type EBV gp42. The sequences of a wild type EBV gp42 protein and a cytoplasmic tail domain of gp42 are provided in Table IB. In some embodiments, the modified EBV gp42s of the disclosure comprise a sequence of any one of SEQ ID NOs: 7-10 or a sequence having at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0490] In some embodiments, the at least one modification relative to a wild type EBV gp42 comprises an insertion, a deletion, a substitution, or a combination thereof.

[0491] In some embodiments, the at least one modification comprises an insertion. In some embodiments, the insertion comprises insertion of a ferritin. The ferritin may be inserted at the C terminal or the N terminal of the modified EBV gp42 protein. In some embodiments, the ferritin comprises a ferritin derived from an amphibian, a bacterium, a fungus, or a plant. In some embodiments, the ferritin comprises a ferritin derived from a bacterium. Nonlimiting examples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H. cetorum, H. vulpis, C. coli, E. coli, andB. subtilis. In some embodiments, the ferritin is derived from E. coli, H. pylori, or P. furiosus. In some embodiments, the ferritin is derived from E. coli. In some embodiments, the ferritin is derived from H. pylori. In some embodiments, the ferritin is derived from P. furiosus. In some embodiments, the ferritin is derived from bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / :. Coli ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / / , pylori ferritin. Exemplary ferritin sequences are provided in Table 4A. In some embodiments, the ferritin comprises an amino acid sequence in Table 4A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ferritin comprises the amino acid sequence of any one of SEQ ID NOS: 440-452 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0492] In some embodiments, the at least one modification comprises a deletion of at least one amino acid residue at the C-terminal, N-terminal, and / or middle portions of the EBV gp42 sequence relative to a wild type gp42. In some embodiments, the deletion comprises deletion of at least 10 amino acids relative to a wild type gp42. In some embodiments, the deletion comprises deletion of at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids,at least 90 amino acids, at least 95 amino acids, or at least 100 amino acids relative to a wild type gp42.

[0493] In some embodiments, the at least one modification comprises substitution of at least one amino acid residue at the C-terminal, N-terminal, and / or middle portions of the EBV gp42 sequence relative to a wild type gp42. In some embodiments, the substitution comprises an amino acid substitution in a gp42 sequence at position 154 relative to SEQ ID NO: 11. In exemplary embodiments, the amino acid substitution at position 154 is arginine (R) to lysine (K), also referred to as “RtoK” herein.

[0494] EBV is known to be associated with multiple sclerosis (MS). The motif that has been identified in numerous viruses that aligns with human peptides and could help explain the prevalence of MS, is a P-(SA)-x-(SGA)-R-(SN)-(LRKH) motif. A similar motif exists in gp42: P-V-V-T-R-N-L. While this peptide is not perfectly synonymous to the motif associated with MS, there is strong sequence homology. Without being held to theory, substituting the critical central arginine residue with a lysine residue may remove this disease-associated epitope and associated risks. In some embodiments of the modified EBV gp42s of the disclosure, the improved characteristic is removal of a disease-associated epitope. In some embodiments of the modified EBV gp42s of the disclosure, the at least one modification comprises an amino acid substitution in a gp42 sequence at position 154 relative to SEQ ID NO: 11 and the improved characteristic is removal of a disease-associated epitope. In exemplary embodiments, the epitope is associated with MS.

[0495] In some embodiments, the modified EBV gp42s of the disclosure comprise a sequence in Table IB or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0496] In some embodiments, the modified EBV gp42s of the disclosure comprise the sequence of any one of SEQ ID NOs: 10-13 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.gH / gL single chain (SC)

[0497] In another aspect, disclosed herein is a modified EBV glycoprotein H / glycoprotein L (gH / gL), wherein the modified EBV gH / gL is a single chain polypeptide comprising an EBV gH amino acid sequence and an EBV gL amino acid sequence, wherein the modified EBV gH / gL comprises at least one modification relative to a wild type EBV gH or gL; andoptionally wherein the modified EBV gH / gL exhibits an improved characteristic relative to a wild type EBV gH or gL.

[0498] In some embodiments, the modified EBV gH / gL is expressed as a heterodimer of gH and gL. In some embodiments, the modified EBV gH / gL comprises a modified EBV gH described herein. In some embodiments, the modified EBV gH / gL comprises a modified EBV gL described herein. In some embodiments, the modified EBV gH / gL comprises a modified EBV gH described herein and an EBV gL. In some embodiments, the modified EBV gH / gL comprises an EBV gH and a modified EBV gL described herein. In some embodiments, the modified EBV gH / gL comprises a modified EBV gH described herein and a modified EBV gL described herein. In some embodiments, the modified EBV gH / gL comprises a sequence of any one of SEQ ID NOs: 1-5 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto and a sequence of SEQ ID NO: 6 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the modified EBV gH / gL comprises a sequence of any one of SEQ ID NOs: 1-5 and a sequence of SEQ ID NO: 6.

[0499] Modifications to each of gH and gL are described above, and the modified EBV gH / gL described herein comprising a single chain polypeptide of gH / gL may comprise one or more of these modifications described above. In some embodiments, the at least one modification relative to a wild type EBV gH or gL comprises an insertion, a deletion, a substitution, or a combination thereof. In some embodiments, the insertion comprises an amino acid insertion of an ERD, an EPM, an ESCRT -independent eVLP inducing domain, a signal peptide, a peptide tag, or a linker. In some embodiments, the insertion comprises insertion of an ERD. In some embodiments, the insertion comprises insertion of an EPM. In some embodiments, the insertion comprises insertion of an ERD and an EPM. The ERD, EPM, ESCRT-independent eVLP inducing domain, signal peptide, peptide tag, or linker may be inserted in any order.

[0500] In exemplary embodiments, the EPM is N-terminal to the ERD or ESCRT-independent eVLP inducing domain. In exemplary embodiments, the ERD or ESCRT-independent eVLP inducing domain is C-terminal to the EPM.

[0501] In some embodiments, the ERD comprises a sequence in Table 2D or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In someembodiments, the ERD comprises the amino acid sequence of any one of SEQ ID NOS:70-108 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0502] In some embodiments, the EPM is derived from a Fc gamma receptor II (FcgR-II). In certain embodiments, the EPM is derived from mouse, common degu, alpine marmot, or golden spiny mouse FcgR-II. In some embodiments, the EPM comprises an amino acid sequence in Table 2C or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the EPM comprises the amino acid sequence of any one of SEQ ID NOS: 60-66 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0503] In some embodiments, the insertion comprises an ESCRT-independent eVLP inducing domain. Non-limiting examples of ESCRT-independent eVLP inducing domains include amphipathic helices, tetraspanins, and the platelet-derived growth factor receptor transmembrane domain. In some embodiments, the ESCRT-independent eVLP inducing domain comprises a sequence in Table 2J or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ESCRT-independent eVLP inducing domain comprises the amino acid sequence of any one of SEQ ID NOS: 500-504 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0504] In some embodiments, the insertion comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence in Table 2A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOS: 40-47 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0505] Engineered EBV polypeptides or modified EBV gH / gL of the disclosure comprising a single chain polypeptide of gH / gL may comprise an insertion of a ferritin sequence. In some embodiments, an engineered EBV polypeptide of the disclosure comprising a single chain polypeptide of gH / gL comprises an insertion of a ferritin sequence. In some embodiments, amodified EBV gH / gL of the disclosure comprising a single chain polypeptide of gH / gL comprises an insertion of a ferritin sequence. The ferritin sequence may be inserted at the C terminal or the N terminal of the gH or gL protein. In some embodiments, the ferritin comprises a ferritin derived from an amphibian, a bacterium, a fungus, or a plant. In some embodiments, the ferritin comprises a ferritin derived from a bacterium. Non-limiting examples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H. cetorum, H. vulpis, C. coli, E. coli, andB. subtilis. In some embodiments, the ferritin is derived from E. coli, H. pylori, or P. furiosus. In some embodiments, the ferritin is derived from E. coli. In some embodiments, the ferritin is derived from H. pylori. In some embodiments, the ferritin is derived from P. furiosus. In some embodiments, the ferritin is derived from bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / :. Coli ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / / , pylori ferritin. Exemplary ferritin sequences are provided in Table 4A. In some embodiments, the ferritin comprises an amino acid sequence in Table 4A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ferritin comprises the amino acid sequence of any one of SEQ ID NOS: 440-452 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0506] Engineered EBV polypeptides or modified EBV gH / gL of the disclosure comprising a single chain polypeptide of gH / gL may comprise a linker. A linker may be used to fuse gH to gL or to a domain or sequence described above, such as an ERD or EPM. In some embodiments, the linker is a moiety derived from the truncated linker of gp350 to gp220. In some embodiments, the linker comprises a soluble loop peptide motif derived from the EBV antigen BMRF-2. Exemplary linker sequences are provided in Table 2G. In some embodiments, the linker comprises an amino acid sequence in Table 2G or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOS: 180-188 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.gH / gL / gp42 Single Chain (SC)

[0507] In another aspect, disclosed herein is a modified EBV glycoprotein H / glycoprotein L / glycoprotein 42 (gH / gL / gp42), wherein the modified EBV gH / gL / gp42 is a single chain polypeptide comprising an EBV gH amino acid sequence, an EBV gL amino acid sequence, and an EBV gp42 amino acid sequence, wherein the modified EBV gH / gL / gp42 comprises at least one modification relative to a wild type EBV gH, gL, or gp42; and optionally wherein the modified EBV gH / gL / gp42 exhibits an improved characteristic relative to a wild type EBV gH, gL, or gp42. In some embodiments, the modified EBV gH / gL / gp42 is expressed as a heterotrimer. In some embodiments, the modified EBV gH / gL / gp42 is expressed as a heterodimer and a monomer. In some embodiments, the modified EBV gH / gL / gp42 is expressed as a gH / gL heterodimer and a gp42 monomer. In some embodiments, the modified EBV gH / gL / gp42 comprises a modified EBV gH described herein. In some embodiments, the modified EBV gH / gL / gp42 comprises a modified EBV gL described herein. In some embodiments, the modified EBV gH / gL / gp42 comprises a modified EBV gp42 described herein. In some embodiments, the modified EBV gH / gL / gp42 comprises a modified EBV gH described herein, an EBV gL, and a modified gp42 described herein. In some embodiments, the modified EBV gH / gL / gp42 comprises an EBV gH, a modified EBV gL described herein, and a modified gp42 described herein. In some embodiments, the modified EBV gH / gL / gp42 comprises a modified EBV described herein gH, a modified EBV gL described herein, and a modified gp42 described herein.

[0508] In some embodiments, the at least one modification relative to a wild type EBV gH, gL, or gp42 comprises an insertion, a deletion, a substitution, or a combination thereof.Modifications to each of gH, gL, or gp42 are described above, and the engineered EBV polypeptide or modified EBV gH / gL / gp42 described herein comprising a single chain gH / gL / gp42 may comprise one or more of these modifications described above. In some embodiments, the at least one modification is in gp42 and comprises a substitution of arginine to lysine at position 154 relative to SEQ ID NO: 11. The substitution of arginine to lysine was made within a position of gp42 that has a sequence that is similar, but not identical, to an identified MS-associated motif. Without being held to theory or mechanism, the amino acid substitution is intended to remove the conserved arginine residue from that motif, thereby removing a MS-associated epitope.

[0509] In some embodiments, the at least one modification comprises an insertion. In some embodiments, the insertion comprises insertion of an ERD, an EPM, an ESCRT-independent eVLP inducing domain, a signal peptide, a peptide tag, a linker, or a ribosomal skip site. Insome embodiments, the insertion comprises insertion of an ERD. In some embodiments, the insertion comprises insertion of an EPM. In some embodiments, the insertion comprises insertion of an ERD and an EPM.

[0510] The ERD, EPM, ESCRT-independent eVLP inducing domain, linker, and / or ribosomal skip site may be inserted in any order. In exemplary embodiments, the EPM is N-terminal to the ERD or ESCRT-independent eVLP inducing domain. In exemplary embodiments, the ERD or ESCRT-independent eVLP inducing domain is C-terminal to the EPM. In exemplary embodiments, the ribosomal skip site is C-terminal to gH and / or gL and is N-terminal to gp42. In exemplary embodiments, the signal peptide is C-terminal to the ribosomal skip site. In exemplary embodiments, the signal peptide is at the N-terminus of the modified EBV gH / gL / gp42.

[0511] In some embodiments, the ERD comprises a sequence in Table 2D or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ERD comprises the amino acid sequence of any one of SEQ ID NOS:70-108 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0512] In some embodiments, the EPM is derived from a Fc gamma receptor II (FcgR-II). In certain embodiments, the EPM is derived from mouse, common degu, alpine marmot, or golden spiny mouse FcgR-II. In some embodiments, the EPM comprises an amino acid sequence in Table 2C or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the EPM comprises the amino acid sequence of any one of SEQ ID NOS: 60-66 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0513] In some embodiments, the insertion comprises an ESCRT-independent eVLP inducing domain. Non-limiting examples of ESCRT-independent eVLP inducing domains include amphipathic helices and the platelet-derived growth factor receptor transmembrane domain. In some embodiments, the ESCRT-independent eVLP inducing domain comprises a sequence in Table 2J or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ESCRT-independent eVLP inducingdomain comprises the amino acid sequence of any one of SEQ ID NOS: 500-504 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0514] In some embodiments, the insertion comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence in Table 2A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOS: 40-47 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0515] In some embodiments, the insertion comprises a ribosomal skip site. In some embodiments, the ribosomal skip site comprises a 2A peptide. The ribosomal skip sites, also referred to as ribosome stop / go sites, are specific sequences that promote ribosomal skipping during protein translation. During a ribosomal skipping event, the ribosome will transiently fall off the growing polypeptide chain (stop) and subsequently resume translation, liberating the first peptide chain, and resulting, assuming successful stop / go activity, in two polypeptide chains without a stop or start codon between them. If the skip site fails, the ribosome reads through the 2A peptide site and the polypeptide is produced as a single chain. Ribosomal skip sites can be derived from different viral species. Different ribosomal skip sites can have different probabilities of resulting in a stop / go event and can therefore be used to control the ratio of ribosomal skipping to read-through.

[0516] In some embodiments, the 2A peptide is derived from foot-and-mouth disease virus, equine rhinitis virus, porcine teschovirus-1, or Thosea asigna virus. The 2A peptide derived from foot-and-mouth disease virus is referred to as F2A. The 2A peptide derived from equine rhinitis A virus is referred to as E2A. The 2A peptide derived from porcine teschovirus-1 is referred to as P2A. The 2A peptide derived from Thosea asigna virus is referred to as T2A. In some embodiments, a GSG linker sequence is added to the 2A peptide. The linker may enhance the ribosome stop / go efficiency at the 2A site. Exemplary 2A peptide sequences are provided in Table 2B below. In some embodiments, the 2A peptide comprises a sequence in Table 2B or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the 2A peptide comprises the amino acid sequence or any one of SEQ ID NOS: 50-57 or a sequence having at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0517] Without being held to theory or mechanism, during ribosomal read-through, the 2A peptide is spontaneously cleaved. This “skipping” occurs only a fraction of the time, depending on the efficiency of the specific 2A peptide. Therefore, when proper ribosomal stop / go, also referred to as cleavage, occurs in the gHgLgp42 single chain polypeptide, gHgL are secreted to the surface as a single chain, and gp42 is produced as a second full-length antigen which is subsequently secreted. Secreted gp42 is capable of associating with the gHgL complex, forming gHgLgp42 heterotrimers on the surface of the host cell. When ribosomal read-through occurs, gHgLgp42 is expressed as a single protein where gHgL is displayed on the surface of the host cell and gp42 exists on the intracellular portion of the transmembrane domain. As a result, only gHgL heterodimers are displayed on the host cell when read-through occurs. Without being held to theory, it is believed that this dual approach could generate improved antigenicity and / or immunogenicity against both gH / gL heterodimers, which is critical for epithelial cell infection, and gH / gL / gp42 heterotrimers, which is critical for B cell infection.

[0518] In some embodiments, the insertion comprises insertion of a cleavage site. In exemplary embodiments, the cleavage site is a furin cleavage site, also referred to as “furin site” herein. The furin cleavage site is a motif recognized by the protease furin. In general, the furin cleavage site occurs after a basic amino acid. In some embodiments, the furin cleavage occurs after a basic acid amino, such as arginine (R) or Lysine (K). In some embodiments, the furin cleavage site motif is R-X-K / R-R. Exemplary sequences for furin cleavage sites are provided in Table 2H. In some embodiments, the furin cleavage site comprises an amino acid sequence in Table 2H or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the furin cleavage site comprises the amino acid sequence of any one of SEQ ID NOS: 190-195 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0519] Engineered EBV polypeptides or modified EBV gH / gL / gp42 of the disclosure comprising single chain polypeptides of gH / gL / gp42 may comprise an insertion of a ferritin sequence. In some embodiments, an engineered EBV polypeptide of the disclosure comprising a single chain polypeptide of gH / gL / gp42 comprises an insertion of a ferritin sequence. In some embodiments, a modified EBV gH / gL / gp42 of the disclosure comprising asingle chain polypeptide of gH / gL / gp42 comprises an insertion of a ferritin sequence. The ferritin sequence may be inserted at the C terminal or the N terminal of the gH, gL, or gp42 protein. In some embodiments, the ferritin comprises a ferritin derived from an amphibian, a bacterium, a fungus, or a plant. In some embodiments, the ferritin comprises a ferritin derived from a bacterium. Non-limiting examples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H. cetorum, H. vulpis, C. coli, E. coli, andB. subtilis. In some embodiments, the ferritin is derived from E. coli, H. pylori, or P. furiosus. In some embodiments, the ferritin is derived from E. coli. In some embodiments, the ferritin is derived from H. pylori. In some embodiments, the ferritin is derived from P. furiosus. In some embodiments, the ferritin is derived from bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / :. Coli ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / / , pylori ferritin. Exemplary ferritin sequences are provided in Table 4A. In some embodiments, the ferritin comprises an amino acid sequence in Table 4A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ferritin comprises the amino acid sequence of any one of SEQ ID NOS: 440-452 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0520] Engineered EBV polypeptides or modified EBV gH / gL / gp42 of the disclosure comprising a single chain polypeptide of gH / gL / gp42 may comprise a linker. A linker may be used to fuse one of the EBV glycoproteins to another EBV glycoprotein or to a domain or sequence described above, such as an ERD or EPM. In some embodiments, the linker is a moiety derived from the truncated linker of gp350 to gp220. In some embodiments, the linker comprises a soluble loop peptide motif derived from the EBV antigen BMRF-2. Exemplary linker sequences are provided in Table 2G. In some embodiments, the linker comprises an amino acid sequence in Table 2G or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOS: 180-188 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0521] In some embodiments, the engineered EBV polypeptides of the present disclosure comprises, from N terminus to C terminus:

[0522] (A) a signal peptide, EBV gL, a linker, EBV gH, a TM domain, a ribosomal skip site, and EBV gp42;

[0523] (B) a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an ERD, a ribosomal skip site, and EBV gp42;

[0524] (C) a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an EPM, an ERD, a ribosomal skip site, and EBV gp42;

[0525] (D) a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an ERD, a furin cleavage site, a ribosomal skip site, and EBV gp42;

[0526] (E) a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an EPM, an ERD, a furin cleavage site, a ribosomal skip site, and EBV gp42;

[0527] (F) a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, and a TM domain;

[0528] (G) a signal peptide, EBV gp42, a linker, EBV gL, a linker, and EBV gH;

[0529] (H) a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, a TM domain, an EPM, and an ERD;

[0530] (I) a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, an EPM, and an ERD;

[0531] (J) a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, a TM domain, and an ERD; or

[0532] (K) a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, a ferritin.

[0533] EBV gH, EBV gL, and EBV gp42 as used in (A) through (K) above refer to both modified or wildtype gH, gL, and gp42, respectively.

[0534] In some embodiments, the engineered EBV polypeptides of the present disclosure comprises, from N terminus to C terminus:

[0535] (A) a signal peptide, EBV gL, a linker, EBV gH, a ribosomal skip site, and EBV gp42;

[0536] (B) a signal peptide, EBV gL, a linker, EBV, gH, an ERD, a ribosomal skip site, and EBV gp42;

[0537] (C) a signal peptide, EBV gL, a linker, EBV, gH, an EPM, an ERD, a ribosomal skip site, and EBV gp42;

[0538] (D) a signal peptide, EBV gL, a linker, EBV, gH, an ERD, a furin cleavage site, a ribosomal skip site, and EBV gp42;

[0539] (E) a signal peptide, EBV gL, a linker, EBV, gH, an EPM, an ERD, a furin cleavage site, a ribosomal skip site, and EBV gp42;

[0540] (F) a signal peptide, EBV gp42, a linker, EBV gL, a linker, and EBV gH;

[0541] (G) a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, an EPM, and an ERD;

[0542] (H) a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, an EPM, and an ERD;

[0543] (I) a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, and an ERD; or

[0544] (J) a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, and a ferritin.

[0545] EBV gH, EBV gL, and EBV gp42 as used in (A) through (J) above refer to both modified or wildtype gH, gL, and gp42, respectively.

[0546] In some embodiments, the engineered EBV polypeptides of the disclosure comprising a single chain polypeptide of gH / gL / gp42 comprises an amino acid sequence in Table 3B, Table 3E, or Table 31 or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the engineered EBV polypeptides of the disclosure comprising a single chain polypeptide of gH / gL / gp42 comprises the amino acid sequence of any one of SEQ ID NOS: 212-219, 240-250, 300-331, and 430-433 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.BMRF-2

[0547] In another aspect, disclosed herein is a modified EBV BMRF-2, wherein the modified EBV BMRF-2 comprises an EBV BMRF-2 amino acid sequence comprising at least one modification relative to a wild type EBV BMRF-2; and optionally wherein the modified EBV BMRF-2 exhibits an improved characteristic relative to a wild type EBV BMRF-2. In some embodiments, the at least one modification relative to a wild type EBV BMRF-2 comprises an insertion, a deletion, a substitution, or a combination thereof. In some embodiments, the at least one modification comprises an insertion. In some embodiments, the insertion comprises an ERD, an ESCRT-independent eVLP inducing domain, an EPM, a signal peptide, a peptide tag, or a combination thereof. In some embodiments, the insertion comprises an ERD. In some embodiments, the insertion comprises an EPM. In some embodiments, the insertioncomprises insertion of an ERD and an EPM. In some embodiments, the insertion comprises insertion of:

[0548] a. an ERD and a signal peptide

[0549] b. an ERD, an EPM, and a signal peptide;

[0550] c. an ESCRT-independent eVLP inducing domain and a signal peptide; or

[0551] d. an ESCRT-independent eVLP inducing domain, an EPM, and a signal peptide.

[0552] The different domains and components of (a) through (d) may be inserted in any order. In exemplary embodiments, the EPM is N-terminal to the ERD or ESCRT-independent eVLP inducing domain. In exemplary embodiments, the ERD or ESCRT-independent eVLP inducing domain is C-terminal to the EPM.

[0553] In some embodiments, the ERD comprises a sequence in Table 2D or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ERD comprises the amino acid sequence of any one of SEQ ID NOS:70-108 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0554] In some embodiments, the EPM is derived from a Fc gamma receptor II (FcgR-II). In certain embodiments, the EPM is derived from mouse, common degu, alpine marmot, or golden spiny mouse FcgR-II. In some embodiments, the EPM comprises an amino acid sequence in Table 2C or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the EPM comprises the amino acid sequence of any one of SEQ ID NOS: 60-66 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0555] In some embodiments, the insertion comprises an ESCRT-independent eVLP inducing domain. Non-limiting examples of ESCRT-independent eVLP inducing domains include amphipathic helices and the platelet-derived growth factor receptor transmembrane domain. In some embodiments, the ESCRT-independent eVLP inducing domain comprises a sequence in Table 2J or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ESCRT-independent eVLP inducing domain comprises the amino acid sequence of any one of SEQ ID NOS: 500-504 or asequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0556] In some embodiments, the insertion comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence in Table 2A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOS: 40-47 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0557] In some embodiments, the at least one modification comprises a deletion. In some embodiments, the deletion comprises a deletion of at least one amino acid residue at the C-terminal, N-terminal, and / or middle portions of the EBV BMRF-2 sequence relative to a wild type BMRF-2. In some embodiments, the deletion comprises deletion of at least 25 amino acids. In some embodiments, the deletion comprises deletion of at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 95 amino acids, at least 100 amino acids, at least 105 amino acids, at least 110 amino acids, at least 115 amino acids, at least 120 amino acids, at least 125 amino acids, at least 130 amino acids, at least 135 amino acids, at least 140 amino acids, at least 145 amino acids, or at least 150 amino acids relative to a wild type BMRF-2 protein.

[0558] In some embodiments, the at least one modification comprises an amino acid substitution. In some embodiments, the substitution comprises substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 amino acids.

[0559] In some embodiments, the modified EBV BMRF-2s of the disclosure comprise an amino acid sequence in Table 3H or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the modified EBV BMRF-2s of the disclosure comprise the amino acid sequence of SEQ ID NO: 420 or SEQ ID NO: 421 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0560] The modified EBV glycoproteins (modified EBV gB, modified EBV gH, modified EBV gL, modified EBV gp42, modified EBV gp350, modified EBV gH / gL, modified EBV gH / gL / gp42, modified EBV BMRF-2) of the present disclosure may exhibit at least oneimproved characteristic relative to a wild type EBV glycoprotein. The at least one improved characteristic may comprise (a) increased stability, (b) increased antigenicity, (c) increased immunogenicity, (d) increased secretion, (e) increased secretion as enveloped virus-like particles (eVLPs), (f) increased secretion as ferritin nanoparticles, (g) increased retention on the cell surface, (h) increased cell-surface expression, (i) increased exposure of target epitopes, (j) decreased exposure of off-target epitopes, (k) decreased off target immune response, (1) increased number of neutralizing epitopes targeted, (m) ability to express multiple EBV glycoproteins from a single polypeptide, (n) increased control of the rate of expression of the modified EBV glycoproteins, (o) removal of a disease-associated epitope, (p) increased neutralization potency, (q) increased neutralization potency relative to total immunogenicity, (r) increased number of epitopes targeted, (s) improved thermal stability, (t) increased stabilization in a prefusion, prefusion-like, or post-fusion conformation, or a combination thereof. In some embodiments, the at least one improved characteristic comprises removal of a disease-associated epitope. In certain embodiments, the disease-associated epitope is a semi-heterologous disease associated epitope. These improved characteristics are described in further detail herein and demonstrated in the Examples.Ferritin Nanoparticles

[0561] In some embodiments, the engineered EBV polypeptides of the disclosure comprises a ferritin. Ferritin is globular protein found in animals, bacteria, and plants, that acts primarily to control the rate and location of polynuclear Fe(III)2O3 formation through transportation of hydrated iron ions and protons to and from a mineralized core. The globular form of ferritin is made up of monomeric subunit proteins (also referred to as monomeric ferritin subunits), which are polypeptides having a molecular weight of approximately 17-20 kDa. Each monomeric ferritin subunit has the topology of a helix bundle which includes a four antiparallel helix motif, with a fifth shorter helix (the c-terminal helix) lying roughly perpendicular to the long axis of the 4-helix bundle. According to convention, the helices are labeled ‘A, B, C, and D & E, from the N-terminus respectively. The N-terminal sequence lies adjacent to the capsid three-fold axis and extends to the surface, while the E helices pack together at the four-fold axis with the C-terminus extending into the particle core. The consequence of this packing creates two pores on the capsid surface. It is expected that one or both of these pores represent the point by which the hydrated iron diffuses into and out of the capsid. Following production, these monomeric ferritin subunit proteins self-assemble into the globular ferritin protein. Thus, the globular form of ferritin comprises 24 monomeric,ferritin subunit proteins, and has a capsid-like structure having 4-3-2 symmetry. Accordingly, when the engineered EBV polypeptides of the present disclosure comprises a ferritin sequence, the ferritins spontaneously self-assemble into 24-valent nanoparticles displaying a three-fold axis of symmetry for presentation of the one or more EBV protein encoded by the engineered EBV polypeptide. For example, a post fusion gB trimer arrayed on a surface of a ferritin nanoparticle is depicted in FIG. IB and the crystal structure of an exemplary gB ferritin nanoparticle is depicted in FIG. 2.

[0562] The ferritin may be incorporated into the engineered EBV polypeptide by insertion of a ferritin sequence at the C terminal or the N terminal of a modified EBV glycoprotein. The ferritin may comprise a ferritin derived from an amphibian, a bacterium, a fungus, or a plant. In some embodiments, the ferritin comprises a ferritin derived from a bacterium. Nonlimiting examples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H. cetorum, H. vulpis, C. coli, E. coli, andB. subtilis. In some embodiments, the ferritin is derived from H. pylori, U urealycitum, E. coli, M. tuberculosis, P. furiosus, C. tepidum, or V. cholera. In some embodiments, the ferritin is derived from E. coli, H. pylori, or P. furiosus. In some embodiments, the ferritin is derived from E. coli. In some embodiments, the ferritin is derived from H. pylori. In some embodiments, the ferritin is derived from P. furiosus. In some embodiments, the ferritin is derived from bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog-E. Coli ferritin. In some embodiments, the ferritin comprises a hybrid bullfrog- / / . pylori ferritin. Exemplary ferritin sequences are provided in Table 4A. In some embodiments, the ferritin comprises an amino acid sequence in Table 4A or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ferritin comprises the amino acid sequence of any one of SEQ ID NOS: 440-452 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0563] A portion of a sequence of a ferritin subunit may be sufficient for directing selfassembly of monomeric ferritin subunits into ferritin nanoparticles. For example, a portion of H. pylori ferritin, located between amino acids 5-168 of the amino acid sequence of H. pylori ferritin, can direct self-assembly into nanoparticles. Additionally, a ferritin amino acid sequence may comprise artificial glycosylation sites, for examples, artificial N-glycosylationsites, which are engineered by inserting artificial mutations into a ferritin amino acid sequence to create a consensus glycosylation sequence. For example, an artificial N-glycosylation site may be created by introducing a consensus sequence N-X-S / T (where X cannot be a P) in a ferritin amino acid sequence. A consensus glycosylation sequence can be created by artificial substitutions of amino acid residues in a ferritin amino acid sequence. For example, an artificial N-glycosylation site in the sequence DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFL NENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFL QWYVAEQHEEEVLFNDILDKIELIGNENHGLYLADQYVKGIAKSRKS (H. Pylori-Ferritin sequence in Table 4A) can be created by introducing two amino acid substitutions: K to N at a position corresponding to position 75, and E to T at a position corresponding to position 75 relative to SEQ ID NO: 440. In another example, an artificial N-glycosylation site in the H. Pylori-V erritin sequence in Table 4 A can be created by introducing two amino acid substitutions: T to N at a position corresponding to position 67 relative to SEQ ID NO: 440 and I to T at a position corresponding to position 69 relative to SEQ ID NO: 440. In yet another example, an artificial N-glycosylation site in the H. Pylori-V erritin sequence in Table 4A (SEQ ID NO: 440) can be created by introducing two amino acid substitutions: H to N at a position corresponding to position 74 relative to SEQ ID NO: 440 and F to T at a position corresponding to position 76 relative to SEQ ID NO: 440. In one more example, an artificial N-glycosylation site in the H. Pylori- mlm sequence in Table 4A (SEQ ID NO: 440) can be created by introducing two amino acid substitutions: E to N at a position corresponding to position 143 relative to SEQ ID NO: 440 and H to T at a position corresponding to position 145 relative to SEQ ID NO: 440. In some embodiments, the ferritin amino acid sequence may include one or more deletions or substitutions, wherein the one or more deletions or substitutions may eliminate a glycosylation site or may allow for more efficient production, ie, of nanoparticles. For example, in some embodiments, the N at a position corresponding to position 9 relative to SEQ ID NO: 440 is substituted for a Q. In some embodiments, the K at a position corresponding to position 131 relative to SEQ ID NO: 440 is substituted for an N.Enveloped virus-like particles (eVLPs)

[0564] Enveloped virus-like particles are referred to herein as eVLPs. Without being held to theory or mechanism, VLPs (virus-like particles) are multiprotein structures that mimic the organization and conformation of authentic native viruses but are non-infectious because they do not contain any viral genome. Their production can be driven by a viral capsid protein, aviral membrane protein, or specific host cell proteins or domains of host cell proteins that specifically mediate formation. The eVLPs of the present disclosure are non-infectious membraned particles whose production does not require a viral capsid protein and is instead driven by one or more polypeptides comprising an eVLP inducing domain. The eVLP inducing domain may comprise an ESCRT recruiting domain (ERD) or an ESCRT-independent eVLP inducing domain. Exemplary sequences for engineered EBV polypeptides and modified EBV glycoproteins of the present disclosure comprising a domain that drives eVLP formation are provided in Tables 3E, 3F, 3G, and 3H. In some embodiments, the engineered EBV polypeptides or modified EBV glycoproteins of the present disclosure comprise an amino acid sequence in Table 3F, Table 3G, or Table 3H, or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the engineered EBV polypeptides or modified EBV glycoproteins of the present disclosure comprise the amino acid sequence of any one of SEQ ID NOS: 300-331, 340-391, 400-409, and 420-421, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0565] Disclosed herein is an enveloped virus-like particle (eVLP) comprising an engineered EBV polypeptide described herein, a modified EBV gB described herein, a modified EBV gH described herein, a modified EBV gL described herein, a modified EBV gp42 described herein, a modified EBV gp350 described herein, a modified EBV BMRF-2 described herein, a modified EBV gH / gL described herein, a modified EBV gH / gL / gp42 described herein, or a combination thereof. Also disclosed herein is an eVLP displaying on its surface all or a portion thereof of an engineered EBV polypeptide described herein, a modified EBV gB described herein, a modified EBV gH described herein, a modified EBV gL described herein, a modified EBV gp42 described herein, a modified EBV gp350 described herein, a modified EBV BMRF-2 described herein, a modified EBV gH / gL described herein, or a modified EBV gH / gL / gp42 described herein.Cells

[0566] In another aspect, disclosed herein is a cell comprising an engineered EBV polypeptide described herein, a modified EBV gB described herein, a modified EBV gH described herein, a modified EBV gL described herein, a modified EBV gp42 described herein, a modified EBV gp350 described herein, a modified EBV BMRF-2 described herein,a modified EBV gH / gL described herein, a modified EBV gH / gL / gp42 described herein, or a combination thereof. Also disclosed herein is a cell displaying on its surface all or a portion thereof of an engineered EBV polypeptide described herein, a modified EBV gB described herein, a modified EBV gH described herein, a modified EBV gL described herein, a modified EBV gp42 described herein, a modified EBV gp350 described herein, a modified EBV BMRF-2 described herein, a modified EBV gH / gL described herein, or a modified EBV gH / gL / gp42 described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.Polynucleotides

[0567] In another aspect, disclosed herein are polynucleotides that encode the amino acid sequence of the engineered EBV polypeptides, the ferritin nanoparticles, or the eVLP described herein. In some embodiments, the polynucleotide is or comprises DNA. In some embodiments, the polynucleotide is or comprises RNA. In some embodiments, the RNA is non-replicating mRNA or virally derived, self-amplifying RNA. In some exemplary embodiments, the RNA is non-replicating mRNA. In exemplary embodiments, the RNA is virally derived, self-amplifying RNA (samRNA) which encodes not only the engineered EBV polypeptides disclosed herein but also viral replication machinery that enables intracellular RNA amplification. For example, in addition to encoding the engineered EBV polypeptides described herein, the samRNA may also encode a viral replicase derived from Venezuelan Equine Encephalovirus (VEEV) followed by a subgenomic promoter. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is trans-amplifying RNA. In some embodiments, the polynucleotides of the present disclosure comprise an Internal Ribosomal Entry Site (IRES) sequence. In some embodiments where the polynucleotide encodes an engineered EBV polypeptide comprising EBV gH, gL, and gp42, an IRES is inserted between the linked gH / gL sequence and the gp42 sequence. In some embodiment, the IRES sequence comprises the sequence in Table 4B or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiment, the IRES sequence comprises the sequence set forth in SEQ ID NO: 460 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0568] Exemplary polynucleotide sequences are provided in Table 5. In some embodiments, the polynucleotides disclosed herein comprises any one of the sequences in Table 5 or asequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the polynucleotides disclosed herein comprises the sequence of any one of SEQ ID NOS: 470-493 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0569] In some embodiments, the polynucleotides of the present disclosure comprise a promoter. In some embodiments, the promoter comprises a ubiquitous promoter, an inducible promoter, a tissue-specific promoter and / or a lineage-specific promoter. In some embodiments, the ubiquitous promoter is selected from the group consisting of: a viral simian virus 40 (SV40) (e.g., early or late), a Rous sarcoma virus (RSV) LTR, an RSV promoter, a cytomegalovirus (CMV) immediate early promoter, a CMV promoter, a Moloney murine leukemia virus (MoMLV) LTR promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, HS, P7. S, and Pl 1 promoters from vaccinia virus, an elongation factor I-alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein S (HSP AS), heat shock protein 90 kDa beta, member 1 (HSP90B 1 ), heat shock protein 70 kDa (HSP70), P-kinesin (P-KIN), the human ROSA 26 locus, a Ubiquitin C promoter (UBC), a phosphoglycerate kinase-I (PGK) promoter, 3 -phosphoglycerate kinase promoter, a cytomegalovirus enhancer, human P-actin (HBA) promoter, chicken P-actin (CBA) promoter, a CAG promoter, a CASI promoter, a CBH promoter, or any combination thereof.

[0570] In some embodiments, the polynucleotides of the present disclosure are operably linked to a tandem gene expression element (e.g., an internal ribosomal entry site (IRES), 2A peptide F2A, E2A, P2A or T2A, poly A tails, or any combination thereof). In some embodiments, the polynucleotide comprises a transcript stabilization element (e.g., woodchuck hepatitis post-translational regulatory element (WPRE), bovine growth hormone polyadenylation (bGH-polyA) signal sequence, human growth hormone polyadenylation (hGH-polyA) signal sequence, or any combination thereof).

[0571] In some embodiments, the polynucleotide of the present disclosure comprises a poly-A tail. Inclusion of a 3’ poly (A) tail in an mRNA sequence can contribute to the stability and translation efficiency of the mRNA. Generally, longer poly(A) tails are associated with increased mRNA stability, thereby allowing their translation and promoting high protein expression.

[0572] In some embodiments, where the polynucleotides of the present disclosure comprise mRNA, the mRNA comprises a poly(A) sequence having at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides. In some embodiments, the poly(A) sequence comprises a short sequence that does not contain alanines in the middle. For example, a poly(A) tail may comprise a long stretch of alanines (e.g., about 60 alanines), followed by a short sequence without alanines, followed by another long stretch of alanines (e.g., about 60 alanines). In some embodiments, the mRNA comprises a 5' untranslated region (UTR), a 3' UTR, and / or a cap. The nucleoside sequence in an mRNA molecule provides instructions that cells use to create specific proteins. mRNA is a molecule that typically is composed of four different nucleosides: adenosine, guanosine, cytidine, and uridine. Vaccines containing mRNA can trigger the body’s own immune system to attack the mRNA molecule. This immune response may destroy the mRNA before it can have its intended effect. Different modifications to the mRNA molecule have been developed to disguise the mRNA from the body’s immune system, such as those described in U. S. Patent No. 10,898,574, U. S. Patent No. 10,703,789, U. S. Patent No. 10,577,403, and U. S. Patent No. 10, 064,959, the contents of which are herein incorporated by reference in their entirety. In some embodiments, where the polynucleotides of the present disclosure comprise mRNA, the mRNA comprises one or more modified nucleotides selected from the group consisting of: pseudouridine, N-l-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,0(6)-methylguanine, and 2-thiocytidine. In some embodiments, the mRNA comprises a modified nucleotide in place of one or more uridines. In some embodiments, the modified nucleoside is selected from pseudouridine (y), N 1-methyl-pseudouridine (m ly), and 5-methyl-uridine(m5U). In some embodiments, the mRNA comprises a modified nucleotide in place of one or more uridines. In some embodiments where the polynucleotides comprise mRNA, the mRNA may be formulated in a lipid nanoparticle (LNP). In some embodiments, the mRNA may be complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, optionally encapsulating mRNA.

[0573] In some embodiments, where the polynucleotide of the present disclosure is mRNA, the mRNA molecule is synthesized through in vitro transcription of a corresponding DNA template molecule. For example, synthetic mRNA can be produced by in vitro transcription of a cDNA template, such as plasmid DNA (pDNA).

[0574] In some embodiments, the sequence of a polynucleotide of the disclosure comprises a modification of at least one nucleotide. In some embodiments, the sequence comprising a modification has at least one improved characteristic relative to an unmodified sequence. In some embodiments, the at least one improved characteristic is improved protein expression. In some embodiments, the at least one improved characteristic is improved protein expression in a mammalian cell. In some embodiments, the at least one improved characteristic is improved mRNA stability. In some embodiments, the at least one improved characteristic is increased content of G and C nucleotides. In some embodiments, the at least one improved characteristic is an improved mRNA folding property. Improved mRNA folding may be determined by comparing mean free energy, with reduced mean free energy corresponding to more structured RNA. Without being held to theory or mechanism, more structured RNA may be easier to translate into protein and may be more stable in solution, thereby increasing protein expression.

[0575] In some embodiments, the polynucleotides of the present disclosure comprise a nucleic acid sequence in Table 5, or a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the polynucleotides of the present disclosure comprise the nucleic acid sequence of any one of SEQ ID NOS: 470-493, or a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the polynucleotides of the present disclosure comprise the nucleic acid sequence of any one of SEQ ID NOS: 480-493, or a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Vectors

[0576] Disclosed herein are vectors comprising the polynucleotide described herein. In some embodiments, the vector is a non-viral vector. Examples of non-viral vectors include, but are not limited to, a plasmid, a transposable element, a naked DNA vector, a lipid nanoparticle (LNP), or any combination thereof. In an exemplary embodiment, the vector is an LNP comprising an mRNA polynucleotide. Generally, LNPs have four components: ionizablelipids, phospholipids, cholesterol, and PEG lipids. Each component contributes to LNP stability, transfection efficacy, and safety.

[0577] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adenovirus, an Adeno-associated virus (AAV), a vesiculovirus, a retrovirus, a herpesvirus, or a vaccinia virus. In some embodiments, the viral vector is an adenovirus. In exemplary embodiments, the adenovirus is a human adenovirus selected from the group consisting of: Adenovirus 3, Adenovirus 5, Adenovirus 26, Adenovirus 35, and Adenovirus 48. In some embodiments, the viral vector is a non-human adenovirus. In exemplary embodiments, the non-human adenovirus is a primate-derived adenovirus, such as a chimpanzee adenovirus. In some embodiments, the viral vector is a vesiculovirus such as Vesicular Stomatitis Virus (VSV). In some embodiments, the viral vector is a retrovirus. In exemplary embodiments, the retrovirus is a lentivirus. In some embodiments, the viral vector is a herpesvirus such as cytomegalovirus (CMV). In some embodiments, the viral vector is a vaccinia virus such as modified vaccinia Ankara (MV A). In some embodiments, the vector is a T7 vector.

[0578] In some embodiments, the polynucleotides and vectors of the present disclosure may be administered to a subject as part of a vaccine composition to prevent or treat a disease or disorder, as will be described in more detail herein.Vaccine Compositions

[0579] Disclosed herein are vaccine compositions comprising the engineered EBV polypeptide described herein, the ferritin nanoparticles described herein, the eVLP described herein, the polynucleotide described herein, or the vector described herein. The vaccine composition described herein may be a subunit vaccine, a ferritin nanoparticle-based vaccine, an eVLP based vaccine, or a nucleic acid vaccine, such as an mRNA vaccine. In some embodiments, the vaccine composition comprises at least two different engineered EBV polypeptides described herein, at least two different polynucleotides described herein; or at least two different vectors described herein.

[0580] In some embodiments, the vaccine composition comprises about 0.1 pg to about lOOpg, or any value or subranges therein, of an engineered EBV polypeptide of the disclosure. In some embodiments, the vaccine composition comprises about 0.1 pg to about 0.5pg, about 0.5pg to about l. Opg, about l. Opg to about 5.0pg, about 5.0pg to about lO. Opg, about lO. Opg to about 20.0 pg, about 20.0pg to about 30.0pg, about 30.0pg to about 40.0pg, about 50.0pg to about 60.0pg, about 60.0pg to about 70.0pg, about 70.0pg to about 80.0pg,about 80. Ogg to about 90. Ogg, or about 90. Ogg to about 100. Ogg of an engineered EBV polypeptide of the disclosure. In some embodiments, the vaccine composition comprises about O.lgg, about 0.2gg, about 0.5gg, about l. Ogg, about 2. Ogg, about 5. Ogg, about 10. Ogg, about 15ug, about 20. Ogg, about 25. Ogg, about 30. Ogg, about 40. Ogg, about 45.0ug about 50. Ogg, about 60. Ogg, about 70. Ogg, about 80. Ogg, about 90. Ogg, or about 100. Ogg of an engineered EBV polypeptide of the disclosure.

[0581] In some embodiments, the vaccine composition comprises about 0.1 pg to about lOOpg, or any value or subranges therein, of a polynucleotide of the disclosure. In some embodiments, the vaccine composition comprises about O.lpg to about 0.5pg, about 0.5gg to about l. Opg, about l. Opg to about 5.0pg, about 5.0pg to about 10. Ogg, about 10. Ogg to about 20. Ogg, about 20.0pg to about 30.0pg, about 30.0pg to about 40. Ogg, about 50. Ogg to about 60. Ogg, about 60.0pg to about 70.0pg, about 70.0pg to about 80. Ogg, about 80.0pg to about 90. Ogg, or about 90. Ogg to about 100. Opg of a polynucleotide of the disclosure. In some embodiments, the vaccine composition comprises about 0.08 gg, about 0.1 gg, about 0.2gg, about 0.4gg, about 0.5gg, about l. Opg, about 1.5gg, about 2. Ogg, about 4. Ogg, about 5. Ogg, about 7.5gg, about 10. Ogg, about 20. Ogg, about 25. Ogg, about 30. Ogg, about 40. Ogg, about 50. Ogg, about 60. Ogg, about 70. Ogg, about 80. Ogg, about 90. Ogg, or about 100. Ogg of a polynucleotide of the disclosure.

[0582] In some embodiments, the vaccine composition comprises a first, a second, and a third engineered EBV polypeptides described herein. In some embodiments, the vaccine composition comprises a first polynucleotide encoding a first engineered EBV polypeptide described herein, a second polynucleotide encoding a second engineered EBV polypeptide described herein, and a third polynucleotide encoding a third engineered EBV polypeptide described herein.

[0583] In some embodiments, the vaccine composition comprises a 1 to 1 to 1 mass ratio of the first, second, and third engineered EBV polypeptides. In some embodiments, the vaccine composition comprises a 5 to 1 to 1 mass ratio of the first, second, and third engineered EBV polypeptides. In some embodiments, the vaccine composition comprises a 1 to 5 to 1 mass ratio of the first, second, and third engineered EBV polypeptides. In some embodiments, the vaccine composition comprises a 1 to 1 to 5 mass ratio of the first, second, and third engineered EBV polypeptides. In some embodiments, the vaccine composition comprises a 1 to 5 to 5 mass ratio of the first, second, and third engineered EBV polypeptides. In some embodiments, the vaccine composition comprises a 5 to 1 to 5 mass ratio of the first, second, and third engineered EBV polypeptides. In some embodiments, the vaccine compositioncomprises a 5 to 5 to 1 mass ratio of the first, second, and third engineered EBV polypeptides.

[0584] In some embodiments, the vaccine composition comprises a 1 to 1 to 1 mass ratio of the first, second, and third polynucleotides. In some embodiments, the vaccine composition comprises a 5 to 1 to 1 mass ratio of the first, second, and third polynucleotides. In some embodiments, the vaccine composition comprises a 1 to 5 to 1 mass ratio of the first, second, and third polynucleotides. In some embodiments, the vaccine composition comprises a 1 to 1 to 5 mass ratio of the first, second, and third polynucleotides. In some embodiments, the vaccine composition comprises a 1 to 5 to 5 mass ratio of the first, second, and third polynucleotides. In some embodiments, the vaccine composition comprises a 5 to 1 to 5 mass ratio of the first, second, and third polynucleotides. In some embodiments, the vaccine composition comprises a 5 to 5 to 1 mass ratio of the first, second, and third polynucleotides.

[0585] In some embodiments, the vaccine composition comprises a 2 to 1 to 1 mass ratio of the first, second, and third polynucleotides. In some embodiments, the vaccine composition comprises a 1 to 2 to 1 mass ratio of the first, second, and third polynucleotides. In some embodiments, the vaccine composition comprises a 1 to 1 to 2 mass ratio of the first, second, and third polynucleotides.

[0586] In some embodiments, after the vaccine composition is delivered to a cell, a glycoprotein or a portion thereof (such as gB) of the first polypeptide is expressed on the surface of an eVLP. In some embodiments, a glycoprotein or a portion thereof (such as gB) of the first polypeptide and a glycoprotein or a portion thereof (such as gp350) of the second polypeptide are expressed on the surface of eVLPs. In some embodiments, a glycoprotein or a portion thereof (such as gB) of the first polypeptide, a glycoprotein or a portion thereof (such as gp350) of the second polypeptide, and a glycoprotein or a portion thereof (such as gH or gL or gH / gL heterodimer or gH / gL / gp42 heterotrimer) of the third polypeptide are expressed on the surface of eVLPs.

[0587] In some embodiments, the vaccine composition further comprises a fourth engineered EBV polypeptide described herein or a fourth polynucleotide encoding a fourth EBV polypeptide described herein. In exemplary embodiments, the fourth engineered EBV polypeptide comprises a modified EBV BMRF-2 glycoprotein. In some embodiments, BMRF-2 is expressed on the surface of an eVLP after the vaccine composition is delivered to a cell.

[0588] In some embodiments where the vaccine composition comprises a first polynucleotide encoding a first engineered EBV polypeptide described herein, a second polynucleotideencoding a second engineered EB V polypeptide described herein, and a third polynucleotide encoding a third engineered EBV polypeptide described herein, the first, second, and third polynucleotides are each incorporated into its own vector. In some embodiments where the vaccine composition comprises a first polynucleotide encoding a first engineered EBV polypeptide described herein, a second polynucleotide encoding a second engineered EBV polypeptide described herein, and a third polynucleotide encoding a third engineered EBV polypeptide described herein, the first, second, and third polynucleotides are incorporated into the same vector.

[0589] In some embodiments where the vaccine composition comprises a first polynucleotide encoding a first engineered EBV polypeptide described herein, a second polynucleotide encoding a second engineered EBV polypeptide described herein, and a third polynucleotide encoding a third engineered EBV polypeptide described herein, the first, second, and third polynucleotides are each encapsulated into its own LNP. In some embodiments where the vaccine composition comprises a first polynucleotide encoding a first engineered EBV polypeptide described herein, a second polynucleotide encoding a second engineered EBV polypeptide described herein, and a third polynucleotide encoding a third engineered EBV polypeptide described herein, the first, second, and third polynucleotides are encapsulated into the same LNPs.

[0590] In some embodiments, the vaccine composition is formulated as a subunit vaccine. In certain embodiments, a subunit vaccine comprises a modified EBV glycoprotein comprising a peptide tag. In certain embodiments, a subunit vaccine comprises a modified EBV glycoprotein comprising an amino acid sequence in Table 3A or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, a subunit vaccine comprises a modified EBV glycoprotein comprising the amino acid sequence of any one of SEQ ID NOS: 210-231, or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0591] In some embodiments, the vaccine composition is formulated as a nucleic acid vaccine. In certain embodiments, the nucleic acid vaccine of comprises one or more polynucleotides comprising a sequences in Table 5 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the nucleic acid vaccine comprises one or more polynucleotides comprising the sequence of any one of SEQID NOS: 470-493 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the nucleic acid vaccine comprises one or more polynucleotides encoding the amino acid sequence of any one of SEQ ID NOS: 210-231, 240-250, 270-283, 290-293, 300-331, 340-391, 400-409, 420-421, and 430-433 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the nucleic acid vaccine is an mRNA vaccine.Orthogonal ferritin

[0592] Ferritin nanoparticles (FNP) can be used as a vaccine platform to deliver EBV glycoproteins as antigens, as described above. Without being held to theory, an effective vaccine against EBV, rather prophylactic or therapeutic, may require inducing an immune response to multiple EBV antigens. However, in some instances, the manufacturing of a multicomponent FNP -based vaccine can be laborious and costly. Therefore, FNP -based vaccine may also be encoded and delivered by nucleic acids, such as mRNAs, for a more efficient manufacturing process. Yet, as the formation of ferritin nanoparticles relies on individual ferritin proteins inside the cells to self-assemble into nanoparticles, co-delivering mRNAs that encode different antigens carried by the same ferritin protein may lead to undesired outcomes. For example, ferritin monomers carrying different antigens may coassemble and lead to erroneous antigen presentation on the ferritin nanoparticles. In addition, ferritin monomers bearing heterologous antigens may compete with ferritin monomers that try to assemble with other ferritin monomers that bear the same antigen, leading to reduced assembling efficiency. Therefore, use of an orthogonal ferritin platform as described herein may be advantageous for delivery of FNP -based vaccines by nucleic acid composed of multiple antigens. Using an orthogonal ferritin platform, each antigen can be linked to a different ferritin, which should reduce erroneous co-assembly of heterologous ferritins, in particular when the ferritins used have low amino acid sequence homology and / or when the different ferritin monomers used only interact with monomers comprising the same sequence and do not interact with monomers comprising a sequence that is not the same.

[0593] In some embodiments, the vaccine compositions of the present disclosure comprise at least a first and a second engineered EBV polypeptide described herein, where the first polypeptide comprises a first ferritin and the second polypeptide comprises a second ferritin. In some embodiments, the vaccine compositions of the present disclosure comprise at least afirst and a second modified EB V glycoprotein described herein, where the first modified EBV glycoprotein comprises a first ferritin and the second modified EBV glycoprotein comprises a second ferritin. In the orthogonal ferritin platform, the first and the second ferritin are not the same. The first and second ferritin may comprise a ferritin derived from an amphibian, a bacterium, a fungus, an archaea, or a plant. In some embodiments, the first and the second ferritin comprises a ferritin derived from a bacterium. Non-limiting examples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H. cetorum, H. vulpis, C. coli, E. coli, and B. subtilis. In some embodiments, the first ferritin is derived from E. coli and the second ferritin is derived from H. pylori. In some embodiments, the first and the second ferritin comprise a hybrid bullfrog ferritin, such as a hybrid bull frog- A. Coli ferritin or a hybrid bullfrog- / / , pylori ferritin.

[0594] In some embodiments, the vaccine compositions comprise a first, a second, and a third engineered EBV polypeptides described herein. In exemplary embodiments, the first engineered EBV polypeptide comprises a modified gB, the second engineered EBV polypeptide comprises a modified gp350, and the third engineered EBV polypeptide comprises gH and gL or gH, gL, and gp42. In some embodiments, each of the first, second, and third engineered EBV polypeptide comprises a first, second, and third ferritin, respectively, where the ferritins are not the same. In exemplary embodiments, the vaccine composition comprises a fourth engineered EBV polypeptide. In certain embodiments, each of the first, second, third, and fourth engineered EBV polypeptide comprises a first, second, third, and fourth ferritin, respectively, where the ferritins are not the same.

[0595] In some embodiments, the vaccine compositions comprise a first, a second, and a third modified EBV glycoprotein described herein. In exemplary embodiments, the first modified EBV glycoprotein comprises a modified gB, the second modified EBV glycoprotein comprises a modified gp350, and the third modified EBV glycoprotein comprises gH and gL or gH, gL, and gp42. In some embodiments, each of the first, second, and third modified EBV glycoprotein comprises a first, second, and third ferritin, respectively, where the ferritins are not the same. In exemplary embodiments, the vaccine composition comprises a fourth modified EBV glycoprotein.

[0596] In some embodiments, the vaccine composition comprises a first, a second, and a third polynucleotide described herein. In exemplary embodiments, the first polynucleotide encodes a first engineered EBV polypeptide comprising a modified gB glycoprotein, the second polynucleotide encodes a second engineered EBV polypeptide comprising a modified gp350glycoprotein, and the third polynucleotide encodes a third engineered EBV polypeptide comprising gH and gL glycoproteins or gH, gL, and gp42 glycoproteins. In some embodiments, each of the first, second, and third engineered EBV polypeptide comprises a first, second, and third ferritin, respectively, where the ferritins are not the same. In some embodiments, the vaccine composition comprises a fourth polynucleotide encoding a fourth engineered EBV polypeptide. In certain embodiments, each of the first, second, third, and fourth engineered EBV polypeptide comprises a first, second, third, and fourth ferritin, respectively, where the ferritins are not the same.

[0597] The first, second, third, and / or fourth ferritin may comprise a ferritin derived from an amphibian, a bacterium, a fungus, am archaea, or a plant. In some embodiments, the first, second, third, or fourth ferritin comprise a ferritin derived from a bacterium. Non-limiting examples of bacteria from which a ferritin may be derived include H. pylori, M. tuberculosis, U urealyticum, P. furiosus, V. cholerae, C. tepidum, H. cetorum, H. vulpis, C. coli, E. coli, andB. subtilis. In some embodiments, the first ferritin is derived from E. coli, the second ferritin is derived from H. pylori, and the third ferritin is derived from P. furiosus. In some embodiments, the first, second, or third ferritin comprise a hybrid bullfrog ferritin. For example, the first ferritin may be a hybrid bullfrog-E. Coli ferritin, the second ferritin may be a hybrid bullfrog- / / , pylori ferritin, and the third ferritin may be a P. furiosus ferritin.

[0598] Any set of ferritin homologs or hybrid versions thereof may be used in the orthogonal ferritin platform described herein. When properly assembled, all or a portion of an EBV glycoprotein of the first polypeptide is expressed on the surface of a first ferritin nanoparticle, all or a portion of an EBV glycoprotein of the second polypeptide is expressed on the surface of a second ferritin nanoparticle, and all or a portion of an EBV glycoprotein of the third polypeptide is expressed on the surface of a third ferritin nanoparticle. All or a portion of an EBV glycoprotein of the fourth polypeptide should be expressed on the surface of a fourth ferritin nanoparticle.Adjuvants

[0599] Aluminium salts (“alum”; aluminum hydroxide, aluminum phosphate) and “Adjuvant System 04” (AS04) are two adjuvants used in commercially available vaccines in the United States. Alum is the most commonly used adjuvant in human vaccination. Additional adjuvants have been approved for use in Europe, and many others are being tested in clinical trials. Non-limiting examples of adjuvants include trehalose-6,6'-dimycolate (TDM), muramyl dipeptide (MDP), pluronic block copolymers, alum solution, aluminium hydroxide,ADJUMER® (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4);AVR1DINE™ (propanediamine); BAY R1005™ ((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amide hydroacetate); CALCITRIOL™ (1-alpha,2S-dihydroxy-vitamin D3); calcium phosphate gel; CAP™ (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin- Al-protein-A-D-fragment fusion protein, sub-unit B of the cholera toxin; CRL 1005 (block copolymer Pl 205); cytokine-containing liposomes; DDA (dimethyldioctadecylammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC / alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i) N-acetylglucosaminyl-(Pl-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(bl-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine); imiquimod (l-(2-methylpropyl)-lH-imidazol-4,5-c)quinoline-4-amine);ImmTher™ (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRVs (immunoliposomes prepared from dehydrati on-rehydration vesicles); interferon-gamma; interleukin- Ibeta; interleukin-2; interleukin-7; interleukin- 12;ISCOMS™; ISCOPREP 7.0.3 ™; liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E. coli labile enterotoxin-protoxin); microspheres and microparticles of any composition; MF59™; (squalene-water emulsion); MONTANIDE ISA 51™ (purified incomplete Freund's adjuvant); MONTANIDE ISA 720™ (metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4'-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-GCn-OCH3); MURAPALMITINE™ and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoCln-sn-glyceroldipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISVs (non-ionic surfactant vesicles); PLEURAN™ (P-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid and glycolic acid; microspheres / nanospheres); PLURONIC L121™; PMMA (polymethyl methacrylate);PODDS™ (proteinoid microspheres); polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80); proteincochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON™ (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-lH-imidazo[4,5-c]quinoline-l-ethanol); SAF-1™ (“Syntex adjuvant formulation”); Sendai proteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and 2,6, 10, 15, 19,23-hexamethyl-2,6, 10, 14, 18,22-tetracosahexane); stearoyltyrosine (octadecyltyrosine hydrochloride); Theramid® (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide); Theronyl-MDP (Termurtide™ or [thr 1]-MDP; N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys, in particular aluminium salts, such as Adju-phos, Alhydrogel, Rehydragel; emulsions, including CFA, SAF, IF A, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121, Poloaxmer4010), etc.; liposomes, including Stealth, cochleates, including BIORAL; plant derived adjuvants, including QS21, Quil A, ISCOMATRIX®, ISCOM; adjuvants suitable for co-stimulation including Tomatine, biopolymers, including PLG, PMM, Inulin; microbe derived adjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleic acid sequences, CpG7909, ligands of human TLR 1-10, ligands of murine TLR 1-13, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPI anchor, LNFPIII / Lewis X, antimicrobial peptides, UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable as antagonists including CGRP neuropeptide.

[0600] Immunostimulatory oligonucleotides (such as those including a CpG motif or Poly(LC) can be used as adjuvants (for example, see U. S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; U. S. Pat. Nos. 6,239,116; 6,339,068; 6,406,705; and 6,429,199).Exemplary adjuvants also may include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary biological adjuvants include STING, IL-2, RANTES, GM-CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.Adjuvants can be used in combination with the disclosed compositions.

[0601] In some embodiments, the vaccine compositions described herein comprise an adjuvant selected from the group consisting of: alum, Alhydrogel, QS-21, saponin, MPLA, squalene, Adju-Phos, and TLR agonists. In some embodiments, the adjuvant is a TLR agonist. In some embodiments, the TLR agonist is an agonist of TLR7, TLR8, and / or TLR9. In some embodiments, the adjuvant is Alhydrogel. In some embodiments, the adjuvant isaluminum hydroxide (“alum”). In some embodiments, the adjuvant is a synthetic form of DNA, an oil in water emulsion, or the like. In some embodiments, the adjuvant is aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, or aluminum potassium sulfate. In some embodiments, the TLR-7 / 8 / 9 agonist is an imidazoquinolinone, such as 3M-052 or R-848. In some embodiments, the TLR-7 / 8 / 9 agonist is a synthetic form of DNA including but not limited to cytosine phosphoguanine (CpG). In some embodiments, the adjuvant is monophosphoryl lipid A or an aluminum salt. In some embodiments, at least two adjuvants are used. For example, in some embodiments, alum and CpG are used in combination as adjuvants.

[0602] In some embodiments, the at least one adjuvant comprises a specific concentration within the composition. For example, when the adjuvant is aluminum hydroxide, the composition may comprise about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1000 pg of aluminum hydroxide per dose. In another example where the adjuvant is CpG, the composition may comprise about 1000 pg, about 1250 pg, about...

Claims

CLAIMS1. A modified EB V glycoprotein B (EBV gB), wherein the modified EBV gB comprises an amino acid sequence comprising at least one modification relative to a wild type EBV gB; and optionally wherein the modified EBV gB exhibits an improved characteristic relative to a wild type EBV gB.

2. The modified EBV gB of claim 1, wherein the modified EBV gB is in a post-fusion conformation.

3. The modified EBV gB of claim 1, wherein the modified EBV gB is in a prefusion or prefusion-like conformation.

4. The modified EBV gB of any one of claims 1-3, wherein the at least one modification relative to a wild type EBV gB comprises an amino acid substitution.

5. The modified EBV gB of claim 4, comprising an amino acid substitution at a position corresponding to position 609 relative to SEQ ID NO: 21.

6. The modified EBV gB of claim 5, comprising an amino acid substitution of histidine (H) to glutamine (Q) at a position corresponding to position 609 relative to SEQ ID NO: 21.

7. The modified EBV gB of any one of claims 4-6, comprising an amino acid substitution at one or more of positions 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, R432, H316, D320, or S325, relative to SEQ ID NO: 21.

8. The modified EBV gB of claim 7, comprising amino acid substitutions at positions corresponding to positions 189, L628, A175, and E634 relative to SEQ ID NO: 21.

9. The modified EBV gB of claim 8, wherein the substitutions at positions corresponding to positions 189, L628, A175, and E634 relative to SEQ ID NO: 21 are I89C, L628GCG, A175C, and E634C, respectively.

10. The modified EBV gB of claim 7, wherein the amino acid substitution comprises substitutions at positions corresponding to positions 189, L628, A175, E634, H316, D320, and S325 relative to SEQ ID NO: 21.

11. The modified EBV gB of claim 10, wherein the substitutions at positions corresponding to positions 189, L628, A175, E634, H316, D320, and S325 relative to SEQ ID NO: 21 are I89C, L628GCG, A175C, E634C, H316I, D320Q, and S325L, respectively.

12. The modified EBV gB of claim 7, comprising substitutions at positions corresponding to W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, and R432 relative to SEQ ID NO: 21.

13. The modified EBV gB of claim 12, wherein the substitutions at positions corresponding to positions W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, and R432 relative to SEQ ID NO: 21 are W112H, Y113R, W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, and R432G, respectively.

14. The modified EBV gB of claim 7, wherein the amino acid substitution comprises substitutions at positions corresponding to 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, and R432 relative to SEQ ID NO: 21.

15. The modified EBV gB of claim 14, wherein the substitutions at positions corresponding to positions 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, and R432 relative to SEQ ID NO: 21 are I89C, L628GCG, A175C, E634C, W112H, Y113R, W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, andR432G, respectively.

16. The modified EBV gB of claim 7, comprising substitutions at positions corresponding to 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, R432, H316, D320, and S325 relative to SEQ ID NO: 21.

17. The modified EBV gB of claim 16, wherein the substitutions at positions corresponding to positions 189, L628, A175, E634, W112, Y113, W193, L194, 1195, W196, D220, R428, R429, R430, R431, R432, H316, D320, and S325 relative to SEQ ID NO: 21are I89C, L628GCG, A175C, E634C, W112H, Y113R, W193R, L194V, I195E, W196A, D220E, R428G, R429G, R430S, R431G, R432G, H316I, D320Q, and S325L relative to SEQ ID NO: 21, respectively.

18. The modified EBV gB of any one of claims 4-17, wherein the improved characteristic is removal of a disease-associated epitope.

19. The modified EBV gB of claim 18, wherein the disease-associated epitope is associated with multiple sclerosis (MS).

20. The modified EBV gB of any one of claims 2-19, wherein the improved characteristic is improved stability.

21. The modified EBV gB of any one of claims 1 - 20, wherein the modified EBV gB comprises an epitope of an EBV gB, wherein the epitope is gB domain D-I, gB domain D-II, gB domain D-III, gB domain D-IV, or gB domain D-V.

22. The modified EBV gB of any one of claims 4-21, wherein the modified EBV gB comprises the sequence of SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 340, or a sequence having at least 70% sequence identity thereto.

23. The modified EBV gB of any one of claims 4-21, comprising an amino acid substitution of at least one amino acid within a furin cleavage site.

24. The modified EBV gB of claim 23, comprising an amino acid substitution of at least one amino acid within a furin cleavage site comprising the motif R-X-K / R-R.

25. The modified EBV gB of claim 24, comprising an amino acid substitution of at least one amino acid within a furin cleavage site comprising the amino acid sequence of any one of SEQ ID NOS: 190-195.

26. The modified EBV gB of any one of claims 4-25, comprising an amino acid substitution wherein:a) a native TM domain corresponding to that of a wildtype gB is substituted with a heterologous TM domain; orb) a native TM domain and a native C-terminal domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain.

27. The modified EBV gB of any one of claims 4-25, comprising an amino acid substitution wherein:a) a native membrane proximal external region (MPER) and a native TM domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain; or b) a native membrane proximal external region (MPER), a native TM domain, and a native C-terminal domain corresponding to that of a wildtype gB are substituted with a heterologous TM domain.

28. The modified EBV gB of any one of claims 1-27, wherein the at least one modification relative to a wild type EBV gB comprises an amino acid deletion.

29. The modified EBV gB of claim 28, comprising an amino acid deletion of at least one amino acid within a furin cleavage site.

30. The modified EBV gB of claim 29, comprising an amino acid deletion of at least one amino acid within a furin cleavage site comprising the motif R-X-K / R-R.

31. The modified EBV gB of claim 30, comprising an amino acid deletion of at least one amino acid within a furin cleavage site comprising the amino acid sequence of any one of SEQ IDNOS: 190-195.

32. The modified EBV gB of any one of claims 1-31, wherein the at least one modification relative to a wild type EBV gB comprises an amino acid insertion.

33. The modified EBV gB of claim 32, comprising an amino acid insertion of a heterologous TM domain.

34. The modified EBV gB of claim 33, comprising an insertion of a heterologous TM domain at the C-terminus of an ectodomain of the modified EBV gB, thereby enablingexpression of all or a portion thereof of the modified EBV gB on the surface of a cell in which it is expressed.

35. The modified EBV gB of any one of claims 26-27 and 33-34, wherein the heterologous TM domain is derived from a viral protein or a bacterial protein.

36. The modified EBV gB of claim 35, wherein the heterologous TM domain is derived from EBV gp220, influenza neuraminidase (NA), Zika virus NS2 protein, or measles virus hemagglutinin.

37. The modified EBV gB of any one of claims 26-27 and 33-36, wherein the heterologous TM domain comprises the amino acid sequence of any one of SEQ ID NOS: 120-125, or a sequence having at least 70% sequence identity thereto.

38. The modified EBV gB of any one of claims 26-27 and 33-37, comprising an insertion of a linker at the C terminus, N terminus, or at the C and N terminals of the heterologous TM domain.

39. The modified EBV gB of any one of claims 32-37, comprising an amino acid insertion of a trimerization domain.

40. The modified EBV gB of claim 39, wherein the trimerization domain is derived from the dimeric GCN4 protein of yeast or the foldon trimerization domain of fibritin protein of T4 bacteriophage.

41. The modified EBV gB of claim 39 or 40, wherein the trimerization domain comprises the amino acid sequence of any one of SEQ ID NOS: 170-173, or a sequence having at least 70% sequence identity thereto.

42. A modified EBV glycoprotein 350 (EBV gp350), wherein the modified EBV gp350 comprises an EBV gp350 amino acid sequence comprising at least one modification relative to a wild type EBV; and optionally wherein the modified EBV gp350 exhibits an improved characteristic relative to a wild type EBV gp350.

43. The modified EBV gp350 of claim 42, wherein the modified EBV gp350 comprises any one of SEQ ID NOS: 30-36, or a sequence having at least 70% sequence identity thereto.

44. A modified EB V glycoprotein H (EBV gH), wherein the modified EB V gH comprises an EBV gH amino acid sequence comprising at least one modification relative to a wild type EBV gH; and optionally wherein the modified EBV gH exhibits an improved characteristic relative to a wild type EBV gH.

45. The modified EBV gH of claim 44, wherein the modified EBV gH comprises an epitope of EBV gH protein, wherein the epitope is gH domain D-I, gH domain D-II, gH domain D-III, or gH domain D-IV.

46. The modified EBV gH of claim 44 or 45, wherein the modified EBV gH comprises the amino acid sequence of any one of SEQ ID NOS: 1-5, or a sequence having at least 70% sequence identity thereto.

47. A modified EBV glycoprotein L (EBV gL), wherein the modified EBV gL comprises an EBV gL amino acid sequence comprising at least one modification relative to a wild type EBV gL; and optionally wherein the modified EBV gL exhibits an improved characteristic relative to a wild type EBV gL.

48. The modified EBV gL of claim 47, wherein the modified EBV gL comprises the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 70% sequence identity thereto.

49. A modified EBV glycoprotein 42 (EBV gp42), wherein the modified EBV gp42 comprises an EBV gp42 amino acid sequence comprising at least one modification relative to a wild type EBV gp42; and optionally wherein the modified EBV gp42 exhibits an improved characteristic relative to a wild type EBV gp42.

50. The modified EBV gp42 of claim 49, wherein the at least one modification comprises an amino acid substitution.

51. The modified EBV gp42 of claim 50, wherein the amino acid substitution is at a position corresponding to position 154 relative to SEQ ID NO: 11.

52. The modified EBV gp42 of claim 51, wherein the amino acid substitution is arginine (R) to lysine (K).

53. The modified EBV gp42 of claim 51 or 52, wherein the improved characteristic is removal of a disease-associated epitope.

54. The modified EBV gp42 of claim 53, wherein the disease-associated epitope is associated with multiple sclerosis.

55. A modified EBV glycoprotein H / glycoprotein L (EBV gH / gL), wherein the modified EBV gH / gL is a single chain polypeptide comprising an EBV gH amino acid sequence and an EBV gL amino acid sequence, wherein the modified EBV gH / gL comprises at least one modification relative to a wild type EBV gH or gL; and optionally wherein the modified EBV gH / gL exhibits an improved characteristic relative to a wild type EBV gH or gL.

56. The modified EBV gH / gL of claim 55, comprising the modified EBV gH of any one of claims 44-46 and / or the modified EBV gL of claim 47 or 48.

57. A modified EBV glycoprotein H / glycoprotein L / glycoprotein gp42 (EBV gH / gL / gp42), wherein the modified EBV gH / gL / gp42 is a single chain polypeptide comprising an EBV gH amino acid sequence, an EBV gL amino acid sequence, and an EBV gp42 amino acid sequence, wherein the modified EBV gH / gL / gp42 comprises at least one modification relative to a wild type EBV gH, gL, or gp42; and optionally wherein the modified EBV gH / gL / gp42 exhibits an improved characteristic relative to a wild type EBV gH, gL, or gp42.

58. The modified EBV gH / gL / gp42 of claim 57, comprising the modified EBV gH of any one of claims 44-46, the modified EBV gL of claim 47 or 48, and / or the modified EBV gp42 of any one of claims 49-54, optionally wherein the at least one modification relative to a wild type EBV gH, gL, or gp42 comprises an amino acid insertion of a ribosomal skip site.

59. A modified EBV glycoprotein BMRF-2, wherein the modified EBV BMRF-2 comprises an EBV BMRF-2 amino acid sequence comprising at least one modification relative to a wild type EBV BMRF-2; and optionally wherein the modified EBV BMRF-2 exhibits an improved characteristic relative to a wild type EBV BMRF-2.

60. The modified EBV BMRF-2 of claim 59, wherein the modified EBV BMRF-2 comprises the amino acid sequence of SEQ ID NO: 421, or a sequence having at least 70% sequence identity thereto.

61. An engineered EBV polypeptide comprising at least one modified EBV glycoprotein, wherein the modified EBV glycoprotein comprises at least one modification relative to a wild type EBV glycoprotein, and optionally wherein the engineered polypeptide exhibits at least one improved characteristic.

62. The engineered EBV polypeptide of claim 61, wherein the at least one modified EBV glycoprotein comprises:a. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of any one of claims 55-56;g. the modified EBV gH / gL / gp42 of any one of claims 57-58; orh. the modified EBV BMRF-2 of claim 59 or 60.

63. The engineered EBV polypeptide of claim 62, wherein the improved characteristic is one or more of: (a) increased stability, (b) increased antigenicity, (c) increased immunogenicity, (d) increased secretion, (e) increased secretion as enveloped virus-like particles (eVLPs), (f) increased secretion as ferritin nanoparticles, (g) increased retention on the cell surface, (h) increased cell-surface expression, (i) increased exposure of target epitopes, (j) decreased exposure of off-target epitopes, (k) decreased off target immune response, (1) increased number of neutralizing epitopes targeted, (m) ability to express multiple EBV glycoproteins from a single polypeptide, (n) increased control of the rate of expression of the modified EBV glycoproteins, (o) removal of a semi-heterologous disease-associated epitope, (p) increased neutralization potency, (q) increased neutralization potency relative to total immunogenicity, (r) increased number of epitopes targeted, or a combination thereof.

64. The engineered EBV polypeptide of claim 62 or 63, wherein the at least one modification comprises an amino acid deletion, an amino acid insertion, or an amino acid substitution.

65. The engineered EBV polypeptide of claim 64, wherein the at least one modification comprises an amino acid deletion.

66. The engineered EBV polypeptide of claim 65, wherein the amino acid deletion comprises a truncation at the C-terminal or N-terminal relative to the amino acid sequence of a wild type glycoprotein.

67. The engineered EBV polypeptide of any one of claims 64 - 66, wherein the at least one modification comprises an amino acid substitution.

68. The engineered EBV polypeptide of any one of claims 64 - 67, comprising two or more consecutive amino acid substitutions.

69. The engineered EBV polypeptide of claim 68, wherein the two or more consecutive amino acid substitution eliminate a furin cleavage site within the engineered EBV polypeptide.

70. The engineered EBV polypeptide of any one of claims 64 - 69, wherein the at least one modification comprises an amino acid insertion.

71. The engineered EBV polypeptide of claim 70, wherein the amino acid insertion comprises insertion of a ribosomal skip site, an endocytosis prevention motif (EPM), a signal peptide, an endosomal sorting complex required for transport (ESCRT) recruiting domain (ERD), an ESCRT-independent eVLP inducing domain, a transmembrane (TM) domain, a trimerization domain, a ferritin sequence, a flexible linker, a cleavage site, a furin cleavage site, an EBV T-cell epitope, or a combination thereof.

72. The engineered EBV polypeptide of claim 71, wherein the engineered EBV polypeptide comprises the modified EBV gH / gL / gp42 of claim 57 or 58, and wherein the amino acid insertion comprises a ribosomal skip site.

73. The engineered EBV polypeptide of claim 72, wherein the ribosomal skip site is a 2A peptide.

74. The engineered EBV polypeptide of claim 73, wherein the 2A peptide is derived from foot-and-mouth disease virus, equine rhinitis virus, porcine teschovirus-1, or Thosea asigna virus.

75. The engineered EBV polypeptide of claim 74, comprising the amino acid sequence of any one of SEQ ID NOS: 50-53 or a sequence having at least 70% sequence identity thereto.

76. The engineered EBV polypeptide of any one of 72-75, wherein the ribosomal skip site is C-terminal to the gH and / or gL sequences and is N-terminal to the gp42 sequence.

77. The engineered EBV polypeptide of any one of claims 71 - 75, wherein the amino acid insertion comprises an EPM.

78. The engineered EBV polypeptide of claim 77, wherein the EPM comprises the amino acid sequence of any one of SEQ ID NOS: 60-66 or a sequence having at least 70% sequence identity thereto.

79. The engineered EBV polypeptide of any one of claims 71 - 78, wherein the amino acid insertion comprises an ERD, and wherein the ERD isa. derived from the ESCRT and ALIX binding region (EABR) of the CEP55 protein or functional analogs from other vertebrate species;b. derived from a viral protein or portion thereof, optionally wherein the viral protein is derived from simian virus 5 (SV) also known as parainfluenza virus 2, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), human T-lymphotropic virus type 1 (HTLV-1), murine leukemia virus(MLV), or Mason-Pfizer monkey virus (MPMV), tick-borne encephalitis virus (TBEV), or a combination thereof; orc. an engineered ERD sequence capable of binding to at least one ESCRT protein.

80. The engineered EBV polypeptide of claim 79, wherein the ERD is derived from an engineered ERD sequence capable of binding to at least one ESCRT protein.

81. The engineered EBV polypeptide of claim 80, wherein the at least one ESCRT protein is ALIX or TSG101.

82. The engineered EBV polypeptide of claim 79, wherein the ERD comprises the amino acid sequence of any one of SEQ ID NOS: 70-108 or a sequence having at least 70% sequence identify thereto.

83. The engineered EBV polypeptide of any one of claims 71 - 82, wherein the amino acid insertion comprises a signal peptide.

84. The engineered EBV polypeptide of claim 83, wherein the signal peptide comprises the signal peptide from IgE, IGVH, tissue plasminogen activator (tPA), CD5, IGKV, or albumin.

85. The engineered EBV polypeptide of claim 84, wherein the signal peptide comprises the amino acid sequence of any one of SEQ ID NOS: 40-47 or a sequence having at least 70% sequence identity thereto.

86. The engineered EBV polypeptide of any one of claims 71 - 85, wherein the amino acid insertion comprises insertion of a transmembrane domain.

87. The engineered EBV polypeptide of claim 86, wherein insertion of the transmembrane domaina. stabilizes the conformation of the modified EBV glycoprotein or a portion thereof;b. anchors the modified EBV glycoprotein or a portion thereof to a membrane when the polypeptide is expressed in a cell; orc. reorients the modified EBV glycoprotein, thereby increasing or decreasing exposure of at least one epitope of the glycoprotein.

88. The engineered EBV polypeptide of claim 87, wherein insertion of the transmembrane domain stabilizes the conformation of the engineered EBV polypeptide or a portion thereof.

89. The engineered EBV polypeptide of claim 88, wherein the TM domain is derived from EBV proteins, Influenza Virus proteins, synthetic TM domains, TM domains from bacteria, TM domains from viruses, and other non-human proteins with 2-pass TM domains.

90. The engineered EBV polypeptide of any one of claims 87-89, wherein insertion of the transmembrane domain anchors the engineered EBV polypeptide or a portion thereof to a membrane when the polypeptide is expressed in a cell.

91. The engineered EBV polypeptide of any one of claims 88-90, wherein the TM domain is inserted in a position that is not naturally anchored to a membrane.

92. The engineered EBV polypeptide of any one of claims 87-91, wherein the TM domain is derived from EBV gp220, influenza neuraminidase (NA), Zika virus NS2 protein, or measles virus hemagglutinin.

93. The engineered EBV polypeptide of any one of claims 86-92, comprising a modified EBV gB comprising a TM domain insertion, wherein the TM domain is inserted at the C-terminus of an ectodomain of the modified EBV gB.

94. The engineered EBV polypeptide of any one of claims 86-92, comprising a modified EBV gB comprising a TM domain insertion, wherein the TM domain is inserted at any one of the amino acid positions selected from: 1-25, 111-114, 160-162, 193-197, 545-547, and 563-565 relative to SEQ ID NO: 20.

95. The engineered EBV polypeptide of any one of claims 86 - 94, comprising the sequences of any one of SEQ ID NOs: 350-391 or a sequence having at least 70% sequence identity thereto.

96. The engineered EBV polypeptide of any one of claims 71 - 95, wherein the amino acid insertion comprises a trimerization domain.

97. The engineered EBV polypeptide of claim 96, wherein the trimerization domain is derived from yeast transcription factor GCN4 or a bacteriophage T4 fibritin foldon trimerization domain.

98. The engineered EBV polypeptide of claim 97, wherein the trimerization domain is modified.

99. The engineered EBV polypeptide of any one of claims 96 - 98, wherein the trimerization domain comprises the amino acid sequence of any one of SEQ ID NOS: 170- 173 or a sequence having at least 70% sequence identity thereto.

100. The engineered EBV polypeptide of any one of claims 71- 99, wherein the amino acid insertion comprises a ferritin.

101. The engineered EBV polypeptide of claim 100, wherein the ferritin comprises a ferritin derived from an amphibian, an archaea, a mammal, a bacterium, a fungus, or a plant.

102. The engineered EBV polypeptide of claim 101, wherein the ferritin comprises a ferritin derived from a bacterium.

103. The engineered EBV polypeptide of claim 102, wherein the bacterium is H. pylori, U urealycitum, E. coli, M. tuberculosis, P furiosus, C. tepidum, or V. cholera.

104. The engineered EBV polypeptide of claim 103, wherein the bacterium is E. coli, H. pylori, or P furiosus.

105. The engineered EBV polypeptide of claim 101, wherein the ferritin comprises a hybrid bullfrog ferritin, wherein the hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from H. pylori, U urealycitum, E. coli, M. tuberculosis, P furiosus, C. tepidum, or V. cholera, or a portion thereof106. The engineered EBV polypeptide of claim 105, wherein the hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from E. coli or a portion thereof.

107. The engineered EBV polypeptide of claim 105, wherein the hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from H. pylori or a portion thereof108. The engineered EBV polypeptide of claim 101, wherein the ferritin comprises the sequence of any one of SEQ ID NOs: 440-452 or a sequence having at least 70% sequence identity thereto.

109. The engineered EBV polypeptide of claim 106, wherein the ferritin comprises the sequence of SEQ ID NO: 452 or a sequence having at least 70% sequence identity thereto.

110. The engineered EBV polypeptide of claim 107, wherein the ferritin comprises the sequence of SEQ ID NO: 440 or a sequence having at least 70% sequence identity thereto.

111. The engineered EBV polypeptide of any one of claims 1 - 110, comprising a peptide tag.

112. The engineered EBV polypeptide of claim 111, wherein the peptide tag is a His-tag, a Strep-tag, Flag-tag, or an Avi-tag.

113. The engineered EBV polypeptide of claim 112, wherein the peptide tag comprises at least six consecutive histidines, or the amino acid sequence of any one of SEQ ID NOS: 200-202 or a sequence with 70% sequence identity thereto.

114. The engineered EBV polypeptide of any one of claims 61 - 113, comprising a peptide linker.

115. The engineered EBV polypeptide of claim 114, wherein the peptide linker is a Gly-Ser linker.

116. The engineered EBV polypeptide of claim 114, wherein the peptide linker is derived from EBVBMRF-2, optionally wherein the peptide linker comprises SEQ ID NO: 180 or a sequence having at least 70% sequence identity thereto.

117. The engineered EBV polypeptide of claim 114, wherein the peptide linker is derived from EBV gp350 or bullfrog ferritin.

118. The engineered EBV polypeptide of any one of claims 114 - 117, wherein the peptide linker is flanked by one, two, or three amino acids on the N-terminus, the C-terminus, or both the N and C terminals.

119. The engineered EBV polypeptide of any one of claims 114 - 118, wherein the peptide linker comprises the amino acid sequence of any one of SEQ ID NOS: 180-188 or a sequence having at least 70% sequence identity thereto.

120. The engineered EBV polypeptide of any one of claims 61 - 119, comprising the amino acid sequence of any one of SEQ ID NOS: 210-231, 240-250, 270-283, 290-293, 300-331, 340-391, 400-409, 420-421, and 430-433 or a sequence having at least 70% sequence identity thereto.

121. The engineered EBV polypeptide of any one of claims 61 - 119, comprising the sequence of any one of SEQ ID NOS: 34, 328, and 388, or a sequence having at least 70% sequence identity thereto.

122. The engineered EBV polypeptide of any one of claims 62 - 119, comprising a modified EBV gH / gL / gp42.

123. The engineered EBV polypeptide of claim 122, comprising an amino acid insertion, wherein the amino acid insertion comprises a signal peptide, a, linker, a ribosomal skip site, a TM domain, an EPM, an ERD, a furin cleavage site, a ferritin, or a combination thereof.

124. The engineered EBV polypeptide of claim 122 or 123, wherein the engineered EBV polypeptide comprises, from N terminus to C terminus:a. a signal peptide, EBV gL, a linker, EBV gH, a TM domain, a ribosomal skip site, and EBV gp42;b. a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an ERD, a ribosomal skip site, and EBV gp42;c. a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an EPM, an ERD, a ribosomal skip site, and EBV gp42;d. a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an ERD, a furin cleavage site, a ribosomal skip site, and EBV gp42;e. a signal peptide, EBV gL, a linker, EBV, gH, a TM domain, an EPM, an ERD, a furin cleavage site, a ribosomal skip site, and EBV gp42;f. a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, and a TM domain;g. a signal peptide, EBV gp42, a linker, EBV gL, a linker, and EBV gH; h. a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, a TM domain, an EPM, and an ERD;i. a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, an EPM, and an ERD;j. a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, a TM domain, and an ERD; ork. a signal peptide, EBV gp42, a linker, EBV gL, a linker, EBV gH, a ferritin.

125. The engineered EBV polypeptide of claim 123 or 124, wherein the linker is a peptide linker derived from EBV BMRF-2 or EBV gp350 or a Gly-Ser linker.

126. The engineered EBV polypeptide of claim 125, wherein the amino acid sequence of the linker comprises any one of SEQ ID NO: 180-188 or a sequence having at least 70% sequence identity thereto.

127. The engineered EBV polypeptide of any one of claims 123 -126, wherein the ribosomal skip site is a 2A peptide.

128. The engineered EBV polypeptide of claim 127, wherein the ribosomal skip site comprises the amino acid sequence of any one of SEQ ID NOS: 50-53 or a sequence having at least 70% sequence identity thereto.

129. The engineered EBV polypeptide of any one of claims 123 - 127, wherein the EPM comprises the amino acid sequence of any one of SEQ ID NOS: 60-66 or a sequence having at least 70% sequence identity thereto.

130. The engineered EBV polypeptide of any one of claims 123 - 129, wherein the TM domain is a single-pass TM domain, a TM domain derived from EBV gp350, or the native TM domain from EBV gH.

131. The engineered EBV polypeptide of any one of claims 123 - 130, wherein the ERD is derived from a viral protein.

132. The engineered EBV polypeptide of claim 131, wherein the viral protein is derived from MPMV, HIV, or a combination thereof, optionally wherein the amino acid sequence of the ERD comprises SEQ ID NO: 37 or SEQ ID NO: 38 or a sequence with 70% sequence identity thereto.

133. An enveloped virus-like particle (eVLP) comprising one or more of:a. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of claim 55 or 56;g. the modified EBV gH / gL / gp42 of claim 57 or 58;h. the modified EBV BMRF-2 of claim 59 or 60; ori. the engineered EBV polypeptide of any one of claims 61 - 132.

134. An enveloped virus-like particle (eVLP) displaying on its surface all or a portion thereof of one or more ofa. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of claim 55 or 56;g. the modified EBV gH / gL / gp42 of claim 57 or 58;h. the modified EBV BMRF-2 of claim 59 or 60; ori. the engineered EBV polypeptide of any one of claims 61 - 132.

135. A cell comprising one or more ofa. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of claim 55 or 56;g. the modified EBV gH / gL / gp42 of claim 57 or 58;h. the modified EBV BMRF-2 of claim 59 or 60; ori. the engineered EBV polypeptide of any one of claims 61 - 132.

136. A cell displaying on its surface all or a portion thereof ofa. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of claim 55 or 56;g. the modified EBV gH / gL / gp42 of claim 57 or 58;h. the modified EBV BMRF-2 of claim 59 or 60; ori. the engineered EBV polypeptide of any one of claims 61 - 132.

137. A polynucleotide encoding the amino acid sequence of:a. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of claim 55 or 56;g. the modified EBV gH / gL / gp42 of claim 57 or 58;h. the modified EBV BMRF-2 of claim 59 or 60; ori. the engineered EBV polypeptide of any one of claims 61 - 132.

138. The polynucleotide of claim 137, wherein the polynucleotide comprises DNA.

139. The polynucleotide of claim 137, wherein the polynucleotide comprises RNA.

140. The polynucleotide of claim 139, wherein the RNA comprises mRNA, selfamplifying RNA, trans-amplifying RNA, or circular RNA.

141. The polynucleotide of claim 140, wherein the RNA comprises modified nucleosides.

142. The polynucleotide of any one of claims 137 - 141, comprising an Internal Ribosomal Entry Site (IRES) sequence.

143. The polynucleotide of claim 142, wherein the IRES sequence comprises the sequence of SEQ ID NO: 182 or a sequence having at least 70% sequence identity thereto.

144. The polynucleotide of any one of claims 137-143, wherein the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NOS: 470-493 or a sequence having at least 70% sequence identity thereto.

145. A vector comprising one or more of the polynucleotides of any one of claims 137-144.

146. The vector of claim 145, wherein the vector is a viral vector.

147. The vector of claim 146, wherein the viral vector is:a. an adenovirus;b. an Adeno-associated virus (AAV);c. a vesiculovirus;d. a retrovirus;e. a herpesvirus; orf. a vaccinia virus.

148. The vector of claim 147, wherein the viral vector is an adenovirus and wherein the adenovirus is a human adenovirus selected from the group consisting of: Adenovirus 3, Adenovirus 5, Adenovirus 26, Adenovirus 35, and Adenovirus 48.

149. The vector of claim 147, wherein the viral vector is a non-human adenovirus, optionally wherein the non-human adenovirus is a primate-derived adenovirus.

150. The vector of claim 149, wherein the non-human adenovirus is a chimpanzee adenovirus.

151. The vector of claim 147, wherein the viral vector is a vesiculovirus and wherein the vesiculovirus is Vesicular Stomatitis Virus (VSV).

152. The vector of claim 147, wherein the viral vector is a retrovirus and wherein the retrovirus is a lentivirus.

153. The vector of claim 147, wherein the viral vector is a herpesvirus and wherein the herpesvirus is cytomegalovirus.

154. The vector of claim 147, wherein the viral vector is a vaccinia virus and wherein the vaccinia virus is modified vaccinia Ankara (MVA).

155. The vector of claim 145, wherein the vector is a non-viral vector.

156. The vector of claim 155, wherein the non-viral vector is a plasmid.

157. The vector of claim 155, wherein the non-viral vector is a lipid nanoparticle (LNP).

158. A vaccine composition comprising:a. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of claim 55 or 56;g. the modified EBV gH / gL / gp42 of claim 57 or 58;h. the modified EBV BMRF-2 of claim 59 or 60;i. the engineered EBV polypeptide of any one of claims 61 - 132;j. the eVLP of claim 133 or 134;k. the polynucleotide of any one of claims 137- 144; orl. the vector of any one of claims 145 - 157.

159. The vaccine composition of claim 158, wherein the composition comprises:a. about O.lug to about lOOug of the engineered EBV polypeptide of any one of claims 1 - 132; orb. about O.lug to about lOOug of the polynucleotide of any one of claims 137— 144.

160. The vaccine composition of claim 158 or 159, comprising:a. at least two different engineered EBV polypeptides selected from any one of claims 1 - 132;b. at least two different polynucleotides selected from any one of claims 137— 144, optionally wherein the at least two different polynucleotides are encapsulated in separate LNPs; orc. at least two different vectors selected from any one of claims 145 - 157.

161. The vaccine composition of claim 160, comprisinga. at least a first and a second engineered EB V polypeptide selected from any one of claims 1 - 132, wherein the first polypeptide comprises a first ferritin and the second polypeptide comprises a second ferritin, and wherein the first and the second ferritin are not the same; orb. at least a first and a second polynucleotide selected from any one of claims 137- 144, wherein the first polynucleotide encodes a first polypeptide comprising a first ferritin and the second polynucleotide encodes a second polypeptide comprising a second ferritin, and wherein the first and the second ferritin are not the same.

162. The vaccine composition of claim 161, wherein the first and the second ferritins are derived from a ferritin of an amphibian, a bacterium, a fungus, an archaea, a mammal, or a plant.

163. The vaccine composition of claim 162, wherein the bacterium is selected from the group consisting of H. Pylori, U Urealycitum, E. Coli, M. Tuberculosis, P Fur iosus, C. Tepidum, and V. Cholera.

164. The vaccine composition of claim 162, wherein the amphibian is a bullfrog.

165. The vaccine composition of any one of claims 161-164, wherein the first or the second ferritin comprises a hybrid bullfrog ferritin, wherein the hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from H. pylori, U urealycitum, E. coli, M. tuberculosis, P furiosus, C. tepidum, or V. cholera, or a portion thereof.

166. The vaccine composition of claim 160, comprising a first and a second engineered EBV polypeptides selected from any one of claims 61 - 132, wherein:a. the first engineered EBV polypeptide comprises gp350; andb. the second engineered EBV polypeptide comprises:i. modified EBV gH and gL;ii. modified EBV gH, gL, and gp42, oriii. modified EBV gH, gL, and gp42; and optionally a linker derived from EBV gp350 or EBV BMRF-2 or a Gly-Ser linker.

167. The vaccine composition of claim 160, comprising a first, a second, and a third engineered EBV polypeptides selected from any one of claims 61 - 132, wherein:a. the first engineered EBV polypeptide comprises a modified EBV gB; b. the second engineered EBV polypeptide comprises gp350; andc. the third engineered EBV polypeptide comprises:i. modified EBV gH and gL;ii. modified EBV gH, gL, and gp42, oriii. modified EBV gH, gL, and gp42; and optionally a linker derived from EBV gp350 or EBV BMRF-2 or a Gly-Ser linker.

168. The vaccine composition of claim 160, comprising a first, a second, and a third polynucleotide selected from any one of claims 137- 144, wherein:a. the first polynucleotide encodes a first engineered EBV polypeptide comprising a modified EBV gB;b. the second polynucleotide encodes a second engineered EBV polypeptide comprising a modified EBV gp350; andc. the third polynucleotide encodes a third engineered EBV polypeptide comprising:i. modified EBV gH and gL;ii. modified EBV gH, gL, and gp42; oriii. modified EBV gH, gL, and gp42 containing a linker derived from EBV gp350 or EBV BMRF-2 or a gly-ser linker.

169. The vaccine composition of claim 167 or 168, wherein:a. a glycoprotein or a portion thereof of the first polypeptide is expressed on the surface of an eVLP;b. a glycoprotein or a portion thereof of the first polypeptide and a glycoprotein of the third polypeptide are expressed on the surface of eVLPs; or c. a glycoprotein or a portion thereof of the first polypeptide, a glycoprotein of the second polypeptide, and a glycoprotein of the third polypeptide are expressed on the surface of eVLPs.

170. The vaccine composition of any one of claims 167 - 169, wherein the first engineered EBV polypeptide comprises a first ferritin and the third engineered EBV polypeptide comprises a second ferritin, and wherein the first and the second ferritin are not the same.

171. The vaccine composition of claim 170, wherein the first and / or the second ferritin comprises a ferritin derived from an amphibian, an archaea, a mammal, a bacterium, a fungus, or a plant.

172. The vaccine composition of claim 171, wherein the first and / or the second ferritin comprises a ferritin derived from a bacterium, optionally wherein the bacterium is H. pylori, U urealycitum, E. coli, M. tuberculosis, P fur iosus, C. tepidum, or V. cholera.

173. The vaccine composition of claim 171, wherein first and / or the second ferritin comprises a ferritin derived from an amphibian, optionally wherein the amphibian is a bullfrog.

174. The vaccine composition of claim 171, wherein the first and / or the second ferritin comprises a hybrid bullfrog ferritin, wherein the hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from H. pylori, U urealycitum, E. coli, M. tuberculosis, P furiosus, C. tepidum, or V. cholera, or a portion thereof.

175. The vaccine composition of any one of claims 170- 174, wherein the second engineered EBV polypeptide comprises a third ferritin and wherein the first, the second, and the third ferritin are not the same.

176. The vaccine composition of any one of claims 167 and 169 - 175, comprising a fourth engineered EBV polypeptide comprising a modified EBVBMRF-2 protein.

177. The vaccine composition of claim 168, comprising a fourth polynucleotide encoding a fourth engineered EBV polypeptide comprising a modified EBV BMRF-2 protein.

178. The vaccine composition of claim 176 or 177, wherein the fourth engineered EBV polypeptide comprises an ERD or ESCRT-independent eVLP inducing domain, and is expressed on the surface of a fourth eVLP179. The vaccine composition of claim 176 or 177, wherein the fourth engineered EBV polypeptide comprises a fourth ferritin, and wherein the first, the second, the third, and the fourth ferritins are not the same.

180. The vaccine composition of claim 168 or 177, wherein the polynucleotides comprise mRNA, self-amplifying RNA, trans-amplifying RNA, or circular RNA.

181. The vaccine composition of any one of claims 158 - 180, comprising at least one adjuvant.

182. The vaccine composition of claim 181, wherein the adjuvant comprises CpG, alum, Alhydrogel, QS-21, saponin, MPLA, squalene, Adju-Phos, a TLR agonist, or a combination thereof.

183. The vaccine composition of claim 182, wherein the TLR agonist is an agonist of TLR7 and / or TLR8.

184. The vaccine composition of any one of claims 158 - 183, comprising one or more pharmaceutically acceptable carrier, excipient, or diluent.

185. A kit comprisinga. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of claim 55 or 56;g. the modified EBV gH / gL / gp42 of claim 57 or 58;h. the modified EBV BMRF-2 of claim 59 or 60;i. the engineered EBV polypeptide of any one of claims 61 - 132; j. the eVLP of claim 133 or 134;k. the polynucleotide of any one of claims 137 - 144;l. the vector of any one of claims 145 - 157; orm. the vaccine composition of any one of claims 158 - 184; and instructions for use.

186. A method of stabilizing EBV gB protein in a prefusion like conformation, wherein the method comprises inserting a heterologous transmembrane domain into the gB protein, whereby the TM domain anchors gB in a prefusion like conformation.

187. A method of expressing an EBV gB protein on the surface of a membrane, wherein the method comprises inserting a heterologous TM domain into one or more sites in the gB protein.

188. The method of claim 187, wherein the TM domain is inserted in anchor site 1, 2, 3, 4, 5, or 6 of gB.

189. The method of claim 187, wherein the TM domain is inserted in a fusion loop of gB.

190. The method of claim 189, wherein the TM domain is inserted in a fusion loop of gB comprising an amino acid sequence of GWYA (SEQ ID NO: 183) or WLIWT (SEQ ID NO: 184).

191. The method of any one of claims 186-190, wherein the TM domain is a two-pass TM domain.

192. The method of claim 191, wherein the two-pass transmembrane domain is derived from Zika virus NS2 protein or influenza virus NA protein.

193. The method of any one of claims 186-192, comprising inserting a linker into the gB protein.

194. The method of claim 193, wherein the linker is a flexible linker.

195. The method of claim 193, wherein the linker is a peptide linker.

196. The method of claim 195, wherein the peptide linker is a Gly-Ser linker.

197. The method of any one of claims 193-196, wherein the linker is directly connected to the N terminus or C terminus of the TM domain.

198. The method of any one of claims 193-197, wherein the length of the linker corresponds to the distance of the insertion site from the membrane.

199. The method of any one of claims 193-198, wherein the length of the linker is between 1 and 40 amino acids.

200. A method of treating an EBV associated cancer or a symptom thereof in a subject having an EBV associated cancer, comprising administering to the subject an effective amount ofa. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of claim 55 or 56;g. the modified EBV gH / gL / gp42 of claim 57 or 58;h. the modified EBV BMRF-2 of claim 59 or 60;i. the engineered EBV polypeptide of any one of claims 61 - 132;j. the eVLP of claim 133 or 134;k. the polynucleotide of any one of claims 137 - 144;l. the vector of any one of claims 145 - 157; orm. the vaccine composition of any one of claims 158 - 184.

201. A method of inducing anti-EBV antibodies and / or inducing a T cell response against EBV in a subject, comprising administering to the subject an effective amount ofa. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of claim 55 or 56;g. the modified EBV gH / gL / gp42 of claim 57 or 58;h. the modified EBV BMRF-2 of claim 59 or 60;i. the engineered EBV polypeptide of any one of claims 61 - 132; j. the eVLP of claim 133 or 134;k. the polynucleotide of any one of claims 137 - 144;l. the vector of any one of claims 145 - 157; orm. the vaccine composition of any one of claims 158 - 184.

202. The method of claim 201, wherein the anti -EBV antibodies bind at least one epitope of EBV gB, gp350, gH, gL, gp42, or BMRF-2.

203. The method of claim 202, wherein the antibodies bind to EBV gB domain D-II or gB domain D-IV.

204. A method for inducing an EBV-specific immune response, comprising administering to the subject an effective amount ofa. the modified EBV gB of any one of claims 1-41;b. the modified EBV gp350 of claim 42 or 43;c. the modified EBV gH of any one of claims 44-46;d. the modified EBV gL of claim 47 or 48;e. the modified EBV gp42 of any one of claims 49-54;f. the modified EBV gH / gL of claim 55 or 56;g. the modified EBV gH / gL / gp42 of claim 57 or 58;h. the modified EBV BMRF-2 of claim 59 or 60;i. the engineered EBV polypeptide of any one of claims 61 - 132; j. the eVLP of claim 133 or 134;k. the polynucleotide of any one of claims 137 - 144;l. the vector of any one of claims 145 - 157; orm. the vaccine composition of any one of claims 158 - 184.

205. The method of any one of claims 200 - 204, wherein the subject is a human.

206. The method of claim 205, wherein the subject is:a. immunosuppressed or immunocompromised;b. an adolescent; orc. under the age of 18.

207. The method of claim 205 or 206, wherein the subject has had a transplant or will have a transplant.

208. The method of any one of claims 200 -207, wherein the subject has a prior EBV infection.

209. The method of any one of claims 200 -208, wherein the subject does not have a prior EBV infection.

210. The method of any one of claims 200 - 209, wherein the route of administration is intramuscular.

211. Use of the modified EBV gB of any one of claims 1-41, the modified EBV gp350 of claim 42 or 43, the modified EBV gH of any one of claims 44-46, the modified EBV gL of claim 47 or 48, the modified EBV gp42 of any one of claims 49-54, the modified EBV gH / gL of claim 55 or 56, the modified EBV gH / gL / gp42 of claim 57 or 58, the modified EBV BMRF-2 of claim 59 or 60, a engineered EBV polypeptide of the disclosure, the eVLP of claim 133 or 134, the polynucleotide of any one of claims 137 - 144, the vector of any one of claims 145 - 157; or the vaccine composition of any one of claims 158 - 184 for manufacture of a medicament for the prevention or treatment of EBV infection or an EBV-associated disease or disorder.

212. Use of the modified EBV gB of any one of claims 1-41, the modified EBV gp350 of claim 42 or 43, the modified EBV gH of any one of claims 44-46, the modified EBV gL of claim 47 or 48, the modified EBV gp42 of any one of claims 49-54, the modified EBV gH / gL of claim 55 or 56, the modified EBV gH / gL / gp42 of claim 57 or 58, the modified EBV BMRF-2 of claim 59 or 60, a engineered EBV polypeptide of the disclosure, the eVLP of claim 133 or 134, the polynucleotide of any one of claims 137 - 144, the vector of any one of claims 145 - 157; or the vaccine composition of any one of claims 158 - 184 for manufacture of a medicament for vaccination against EBV infection.

213. The modified EBV gB of any one of claims 1-41, the modified EBV gp350 of claim 42 or 43, the modified EBV gH of any one of claims 44-46, the modified EBV gL of claim 47 or 48, the modified EBV gp42 of any one of claims 49-54, the modified EBV gH / gL of claim 55 or 56, the modified EBV gH / gL / gp42 of claim 57 or 58, the modified EBV BMRF-2 of claim 59 or 60, a engineered EBV polypeptide of the disclosure, the eVLP of claim 133 or 134, the polynucleotide of any one of claims 137 - 144, the vector of any one of claims 145 -157; or the vaccine composition of any one of claims 158 - 184 for use in the treatment or prevention of EBV infection or an EBV-associated disease or disorder.

214. The modified EBV gB of any one of claims 1-41, the modified EBV gp350 of claim 42 or 43, the modified EBV gH of any one of claims 44-46, the modified EBV gL of claim 47 or 48, the modified EBV gp42 of any one of claims 49-54, the modified EBV gH / gL of claim 55 or 56, the modified EBV gH / gL / gp42 of claim 57 or 58, the modified EBV BMRF-2 of claim 59 or 60, a engineered EBV polypeptide of the disclosure, the eVLP of claim 133 or 134, the polynucleotide of any one of claims 137 - 144, the vector of any one of claims 145 -157; or the vaccine composition of any one of claims 158 - 184 for use in the vaccination against EBV infection.

Images