Prefusion-stabilized EBV GB proteins

Abstract

Provided herein are engineered EBV gB polypeptides. In some aspects, the engineered gB polypeptides exhibit enhanced conformational stability and / or immunogenicity / antigenicity of the prefusion conformation. Methods are also provided for use of the engineered gB polypeptides as diagnostics, in screening platforms, and / or in vaccine compositions.

Description

DESCRIPTIONPREFUSION-STABILIZED EBV GB PROTEINSREFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of United States provisional application number 63 / 805,170, filed May 13, 2025; United States provisional application number 63 / 775,767, filed March 21, 2025; United States provisional application number 63 / 772,502, filed March 15, 2025; and United States provisional application number 63 / 689, 195, filed August 30, 2024, the entire contents of each of which are incorporated herein by reference.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on August 18, 2025, is named UTFBP1372WO_ST26.xml and is 916,263 bytes in size.BACKGROUND1. Field

[0003] The present disclosure relates generally to the fields of medicine, virology, and immunology. More particularly, it concerns engineered Epstein-Barr virus (EBV) glycoprotein B (gB) polypeptides and uses thereof.2. Description of Related Art

[0004] Epstein-Barr virus (EBV) is one of the most common human viruses in the world. EBV is also known as human herpesvirus 4 and is a member of the herpes virus family. Most people will get infected with EBV in their lifetime, especially in childhood, and may not have symptoms. However, if symptoms do present, they usually include fatigue, fever, inflamed throat, swollen lymph nodes in the neck, enlarged spleen, swollen liver, and rash. Usually, only teenagers and adults get symptoms, and the symptoms of acute infection are usually gone in 2 to 4 weeks.

[0005] EBV can lie dormant within a cell indefinitely in a latent infection and not be fully eradicated even after treatment. The result is that the virus can reactivate and begin 4930-2385-3138, v. 1- 1 -4930-2385-3138, v. 1producing large amounts of viral progeny without the host being infected by any new outside virus. In the latent state, the viral genome persists within the host cells as episomes.

[0006] There is no cure for EBV and, once infected, a host may carry the virus indefinitely, even when not presenting symptoms. Because latent infections can evade immune surveillance and reactivate the lytic cycle at any time, there is a persistent risk to an infected individual of outbreak and the pain and suffering associated with it. Additionally, these persistent reactivations of EBV can result in a plurality of associated long-term sequelae including many forms of cancer, including lymphomas, epithelial cancers, as well as neurodegenerative diseases such as multiple sclerosis (MS).

[0007] Accordingly, safe and effective immunogenic and / or antigenic compositions to protect against EBV infection are needed. Diagnostic reagents to detect immune responses to EBV, to guide the design of gB-based EBV vaccines, and to support the development of therapeutic or prophylactic antibodies against EBV are also needed.SUMMARY

[0008] As such, provided herein are engineered proteins having at least one amino acid mutation relative to the amino acid sequence of a wild- type Epstein-Barr virus glycoprotein B (EBV gB) protein. The engineered proteins may be stabilized in the prefusion conformation of EBV gB. The engineered proteins may specifically bind to an EBV gB prefusion-specific antibody.

[0009] Provided herein are engineered proteins comprising engineered EBV gB protein ectodomains comprising a sequence having at least 80% identity, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 22-672 of SEQ ID NO: 1, 74, or 78, said engineered protein comprising at least one substitution or set of substitutions selected from the group consisting of: Q527C / E634C, S652C / A672C, N88C / L628C, A175C / E634C, T498C / F655C, N88C / L628GCG, I89C / L628GCG,N88C / L628XCX (where X is any amino acid), I89C / L628XCX (where X is any amino acid), H316I / D320Q / S325L, and Q460C / I461C, wherein the positions are relative to SEQ ID NO: 1.- 2 -4930-2385-3138, v. 1

[0010] The engineered EBV gB protein ectodomains may comprise a substitution or set of substitutions selected from any one of the substitutions or sets of substitutions of Table 1. The engineered protein may comprise an amino acid sequence having at least 80% identity, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 26-559.

[0011] The engineered EBV gB protein ectodomains may comprise an amino acid sequence having at least 80% identity, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 22-672 of SEQ ID NO: 1, 74, or 78. The engineered EBV gB protein ectodomains may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 amino acid substitutions relative to the sequence of amino acids 22-672 of SEQ ID NO: 1.

[0012] Without being held to theory or mechanism, the engineered EBV gB protein ectodomains may exhibit improved solubility or stability in the prefusion form, as compared to a native gB, which exists mainly in a postfusion conformation. The engineered EBV gB protein ectodomains may be immunogenic and / or antigenic.

[0013] The engineered proteins may or may not comprise an N-terminal signal sequence. The engineered proteins may or may not comprise the membrane proximal region, transmembrane region, and / or cytoplasmic tail of EBV gB.

[0014] The engineered EBV gB protein ectodomains may be fused or conjugated to a trimerization domain. The engineered EBV gB protein ectodomains may be fused to a trimerization domain. The trimerization domain may comprise a T4 fibritin trimerization domain (Fd); a GCN4 domain; a 4J4A domain; an anti-parallel, two-helix bundle comprising complementary first heptad repeat (HR1) and second heptad repeat (HR2) regions; or a combination thereof. In some aspects, the trimerization domain is fused to the C-terminus of the ectodomain via a Gly-Ser linker.- 3 -4930-2385-3138, v. 1

[0015] The engineered EBV gB protein ectodomains may be fused or conjugated to a transmembrane domain. The EBV gB protein ectodomains may be fused to a transmembrane domain. The transmembrane domain may comprise an EBV gB protein transmembrane domain. The transmembrane domain may not comprise an EBV gB protein transmembrane domain.

[0016] The engineered proteins may comprise a sequence, named ESCRT-recruiting domain (ERD), to recruit the endosomal sorting complex required for transport (ESCRT). The ERD may be inserted N-terminally, C-terminally, or internally. These engineered proteins also comprise a transmembrane domain and cytoplasmic domain of EBV gB. The engineered proteins may comprise R837E and R838E substitutions in the EBV gB cytoplasmic domain. The EBV gB protein ectodomain may comprise or further comprise an elimination of the furin cleavage site located at positions 428-432. The amino acids at these positions can each be independently substituted to another amino acid, or deleted, such that the remaining sequence is anything other than RX(K / R)R. For example, the EBV gB protein ectodomain may comprise R428G, R429G, R430S, R431G, and R432G substitutions. The engineered protein may comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 22-967 of SEQ ID NO: 74. The engineered protein may comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 74, 161-163, 257-259, 274, 276, 323-327, 334-337, 385, 404-407, 454, and 455.

[0017] Provided herein are engineered EBV gB protein trimers comprising three engineered ectodomains or engineered proteins as provided herein. The trimers may be stabilized in a prefusion conformation relative to a trimer of wild-type EBV gB protein subunits. The trimers may be immunogenic and / or antigenic.

[0018] Provided herein are nucleic acid molecules comprising a nucleotide sequence that encodes an amino acid sequence of any engineered ectodomain or engineered protein provided herein. The nucleic acid molecule may be a DNA expression vector, an mRNA, a self-replicating RNA, or a viral vector. The self-replicating RNA may be a trans amplifying RNA or a circular RNA. The nucleic acid may comprise at least one chemical modification, such as, for example, a chemical modification selected from the group consisting of- 4 -4930-2385-3138, v. 1pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, N1 -ethylpseudouridine, 2- thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouri dine, 2-thio- 1 -methyl-pseudouridine, 2-thio-5 -aza-uridine , 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l -methyl-pseudouridine, 4-thio-pseudouridine, 5 -aza-uridine, dihydropseudouridine, 5 -methoxy uridine and 2'-0-methyl uridine.

[0019] Provided herein are pharmaceutical compositions comprising (i) an engineered ectodomain or engineered protein provided herein, (ii) an engineered trimer provided herein, or (iii) a nucleic acid molecule provided herein; and a pharmaceutically acceptable carrier. The composition may comprise an adjuvant. The composition may be formulated within a cationic lipid. The composition may further comprise one or more additional EBV glycoproteins, or a nucleic acid molecule encoding such additional EBV glycoprotein(s). The additional EBV glycoproteins may be gH / gL, gp42, and / or gp350.

[0020] Provided herein are methods of eliciting an immune response to EBV in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition provided herein. Provided herein are methods of treating or preventing EBV infection, a reactivation of latent EBV in a patient infected with EBV, or a disease associated with EBV infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0021] Any pharmaceutical composition provided herein is contemplated for use in eliciting an immune response against EBV. Any pharmaceutical composition provided herein is contemplated for use in the prevention of an EBV infection, a reactivation of latent EBV in a patient infected with EBV, or a disease associated with EBV infection in a subject.

[0022] Any (i) an engineered ectodomain or engineered protein provided herein, (ii) an engineered trimer provided herein, or (iii) a nucleic acid molecule provided herein is contemplated to be used in the manufacture of a medicament for the prevention of an EBV infection, a reactivation of latent EBV in a patient infected with EBV, or a disease associated with EBV infection.

[0023] Provided herein are compositions comprising (i) an engineered ectodomain or engineered protein provided herein or (ii) an engineered trimer provided herein bound to an antibody. The antibody may specifically bind to an EBV gB in the prefusion conformation.- 5 -4930-2385-3138, v. 1

[0024] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0026] FIG. 1. Representative non-reducing SDS-PAGE gels for various constructs, stained with Coomassie blue. Sequence numbering indicated at the top of the SDS-PAGE gels and molecule weight (MW, in kDa) shown on left. For designs that utilize interprotomer disulfides the expected molecular weight of seen on a non-reducing gel is that of the trimer, while those designs which do not contain interprotomer disulfides would be expected to run as a monomer.

[0027] FIG. 2. Mass profiles for various constructs as analyzed by mass photometry. LMW: Low molecular weight SEC peak; MMW: medium molecular weight SEC peak; HMW: high molecular weight SEC peak. All traces show a strong peak at the anticipated trimeric molecular weight. In many traces subsequent dimer of trimers, trimer of trimers, etc. peaks are seen - consistent with protein agglomeration.

[0028] FIG. 3. Size exclusion chromatographs for various constructs.

[0029] FIG. 4. Differential scanning fluorimetry (DSF) results for various constructs. DSF detects changes in fluorescence that result from exposure of previously concealed residues upon the changes of protein structure. It is predicted that the prefusion molecule will have a different DSF profile as a result of the existence of an initial melting transition where the prefusion conformation melts. This method, therefore, can be used to rapidly profile different candidate designs.- 6 -4930-2385-3138, v. 1

[0030] FIG. 5. HiTrap Q HP Anion Exchange data for EBV-415.

[0031] FIG. 6. Negative-stain electron microscopy micrographs, 2D classes, and 3D maps.

[0032] FIG. 7. Cryo-EM 2D class averages and 3D maps for various constructs.

[0033] FIG. 8. The structure of EBV-415 with key residues in Domain IV (residues 620-640) and Domains I, II, and IV (residues 53-63, 86-91, 174,175, 212-216, 225-227, 499- 502, 517-518, 525-529) highlighted. Cysteine insertions and / or substitutions allowing for disulfide bond formation between these two regions are involved in stabilization of EBV gB in the prefusion conformation.

[0034] FIGS. 9A-9B. Design theory for EBV gB Base, shaded by domain. (FIG. 9A) Schematic of wildtype (WT) gB and ectodomain base construct (EBV Base). The 22 residue N-terminal signal sequence (SS) and the C-terminal domain (C-term) are shown as white boxes. The schematic is shaded by domain: domain I (DI); DII; Dill; DIV. DV is split into the N- and C-terminal regions. Native disulfide bonds are shown as connecting black lines. The native fusion loops are shown as dashed black boxes. N-linked glycosylation sites are shown as branched lines. The native furin cleavage site is shown as a thick black line. The membrane proximal region (MPR) and transmembrane domain (TM) are shown in grey with the TM marked by dashed blue lines. EBV Base consists of the first 688 residues of WT gB with the fusion loops (WY112 1 13and WLIW193 196) substituted for the corresponding HSV-2 residues (HR177-178and RVEA258-261), which are shown as dashed boxes. The furin cleavage site (RRRRR428-432) was replaced with GGSGG, as shown with a thick line. A D220E substitution from the WT B95-8 strain is also included in EBV Base and is shown as a tan line. Residue 688 is followed by the foldon (Fd) domain and C-terminal tags. (FIG. 9B) Side view of the AlphaFold2 model of prefusion WT gB with signal sequence residues hidden. Trimeric prefusion HCMV gB (PDB ID: 7KDP, Liu Sci. Adv. 2021) was used as a template to produce the prefusion EBV gB model. One protomer is shaded as in (Panel A) and shown as a ribbon diagram, one protomer is shaded gray and shown as a cartoon trace of the a- carbon backbone, and one protomer is shown as a transparent surface. The “elbow” allowing DI to splay out in partially-prefusion stabilized ectodomain constructs is labeled and marked with arrows.- 7 -4930-2385-3138, v. 1

[0035] FIGS. 10A-10E. Characterization of EBV gB variants suggests subtle structural differences from HCMV gB. (FIG. 10A) Schematic of wildtype (WT) gB and ectodomain base construct (EBV Base). EBV Base contains modifications listed in the diagram. (FIG. 10B) AlphaFold2 model of prefusion WT gB. Trimeric prefusion HCMV gB (PDB ID: 7KDP) was used as a template. One protomer is shaded as indicated in (FIG. 10A) and shown as a ribbon, one is shaded gray and shown as a trace of the a-carbon backbone, and one is shown as a transparent surface. Inset shows EBV gB residues homologous to those used to stabilize a prefusion-like HCMV gB ectodomain. (FIG. 10C) Non-reducing SDS- PAGE analysis of gB variants. Molecular weight standards are indicated on the left in kDa. White, grey, and black triangles denote the monomeric, dimeric, and trimeric forms of gB, respectively. (FIG. 10D) DSF analysis of gB variant thermostability, as indicated in (FIG. 10C). (FIG. 10E) nsEM 2D averages of top single-disulfide variants. White arrow highlights a splayed-out DI.

[0036] FIGS. 11 A- 11C. Analysis of EBV gB double-disulfide variants. (FIG. 11 A) Non-reducing SDS-PAGE. (FIG. 1 IB) Differential scanning fluorimetry (DSF) analysis, as indicated in (FIG. 11 A). (FIG. 11 C) nsEM 2D class averages of variants EBV-336 and EBV- 395. The white arrow highlights a splayed-out DI. Scale bar: 130 A

[0037] FIGS. 12A-12G. Characterization of EBV gB combination variants. (FIG. 12A) Non-reducing SDS-PAGE analysis of gB variants. Molecular weight standards are indicated on the left in kDa. Shaded triangles denote the monomeric, dimeric, and trimeric forms of gB. (FIG. 12B) Differential scanning fluorimetry (DSF) analysis of gB variant thermostability, as indicated in (FIG. 12A). (FIG. 12C) Negative-stain electron microscopy (nsEM) of top single-disulfide variants. White arrows highlight a splayed-out DI. Scale bars: 130 A. (FIG. 12D) Binding of a-gB antibodies to EBV-53 (top panel) and EBV-460 (bottom panel) as measured by biolayer interferometry. Neutralizing antibodies are shown as solid lines in shades of green. Non-neutralizing antibodies are shown as dashed lines in shades of black / grey. Vertical dotted lines represent transition from association of antibodies to dissociation. (FIG. 12E) Representative micrograph from cryo-EM data collection for variant EBV-460. Scale bar = 25 nm. (FIG. 12F) Representative 2D class averages of variant EBV- 460. Scale bar = 110 A. (FIG. 12G) 3.1 A cryo-EM map and model of variant EBV-460 shaded by domain as in FIG. 9 panel B, but with the surface representation replaced with the cryo-EM map.- 8 -4930-2385-3138, v. 1

[0038] FIG. 13. I89C / L628(GCG) is involved in stabilizing prefusion gB. (Left panel) Non-reducing SDS-PAGE analysis of gB variants. Molecular weight standards are indicated on the left in kDa. Shaded triangles denote the monomeric, dimeric, and trimeric forms of gB. (Right panel) Differential scanning fluorimetry (DSF) analysis of gB variant thermostability, as indicated in (Left panel).

[0039] FIG. 14. A293P reduces expression of a soluble construct. (Left panel) Nonreducing SDS-PAGE analysis of gB variants. Molecular weight standards are indicated on the left in kDa. (Right panel) Differential scanning fluorimetry (DSF) analysis of gB variant thermostability.

[0040] FIGS. 15A-15D. Results of experiments showing that prefusion gB of the disclosure provides enhanced protection against epithelial cells and B cell infection by EBV. (FIG. 15 A) Schematic of mouse immunization study and serum collection timeline. Female BALB / c mice (7-8 weeks old, n= 5 / group / experiment, 2 independent experiments) were immunized with one of the antigens indicated in the diagram. (FIG. 15B) Binding response presented as the ratio of binding to prefusion gB (Pre-gB; EBV-460) divided by the binding to postfusion gB (Post-gB), normalized to the average ratio of binding observed in sera from mice vaccinated with postfusion gB. Binding was measured by a Luminex assay. Data are plotted as the average of two technical replicates. The mean is shown as a horizontal black line and error bars represent the standard deviation. (FIG. 15C) Neutralization by sera from immunized mice for EBV infection of the indicated cells. Data are plotted for individual mice. The geometric mean is shown as a horizontal black line and the error bars represent the geometric standard deviation. (FIG. 15D) Neutralization by sera from human volunteers. Sera were depleted as indicated on the x-axis. Statistical significance was determined by two-way ANOVA followed by Tukey’s HSD test (FIGS. 15B, 15C) or by paired t-tests (FIG. 15D) performed in GraphPad Prism vlO.4.2: *P < 0.05.

[0041] FIGS. 16A-16D. A 3.1 A cryo-EM structure of D2C3 (EBV-460). (FIG. 16A) Side view of the D2C3 (EBV-460) model. One protomer is shown as a ribbon diagram, one protomer is shown as the opaque cryo-EM map, and one protomer is shown as the transparent cryo-EM map with the ribbon diagram of the model shown underneath. One N-linked glycan chain per protomer was built and is shown as tan sticks. The linked asparagine residue is shown as sticks shaded the same as its respective ribbon diagram. (FIG. 16B-16D) Zoomed views of substitutions that comprise D2C3 (EBV-460). Key residues are shown as sticks.- 9 -4930-2385-3138, v. 1

[0042] FIG. 17. Sequence alignment of EBV gB, HCMV gB, HSV-2 gB, and prefusion EBV gB (D2C3). Multiple sequence alignment of EBV gB (M81 strain, GenBank: AWG92945.1; SEQ ID NO: 560), HCMV gB (Towne strain, GenBank: Pl 3201.1; SEQ ID NO: 561), HSV-2 gB (HG52 strain, GenBank: YP_009137179.1 ; SEQ ID NO: 562), and prefusion EBV gB (D2C3; EBV-460; SEQ ID NO: 563) was performed using Clustal Omega vl.2.4 and formatted with ESPript v3.0. The alignment is limited to residues 42-688, corresponding to the first and last residues resolved for the postfusion and prefusion EBV gB structures, respectively. Gaps in secondary structure correspond to unresolved residues. Select residues and features are marked as indicated in the legend above. Secondary structure elements were derived from the postfusion EBV gB crystal structure (Post-gB, PDB: 3FVC) or from the prefusion EBV gB structure (Pre-gB) and are shaded by domain as in FIG. 9.

[0043] FIGS. 18A-18C. Effects of individual modifications on EBV Base. (FIG. 18A) Non-reducing SDS-PAGE analysis of gB variants. (FIG. 18B) Reducing SDS-PAGE analysis of gB variants. (FIG. 18C) DSF analysis of gB variant thermostability indicated as in (A).

[0044] FIGS. 19A-19C. Alignment of the AlphaFold2 model of prefusion EBV gB with two HCMV gB prefusion structures. (FIG. 19A) The EBV gB prefusion model (AlphaFold2) aligned with membrane-anchored HCMV gB structure (PDB:7KDP). (FIG. 19B) The EBV gB prefusion model aligned with the HCMV gB ectodomain structure (PDB: 8VYM). The black arrow highlights the movement of domain I (DI) between the EBV gB model and the HCMV gB ectodomain structure. (FIG. 19C) Pairwise alignments of DI-DIV between the EBV gB model and the HCMV gB ectodomain structure. The EBV gB model is shaded by domain as in FIG. 9. HCMV gB is indicated as in (FIG. 19B). All alignments were performed using the MatchMaker function in ChimeraX. Individual domains were aligned based on residues within the respective domain.

[0045] FIG. 20. EBV-460 storage stability test. Differential scanning fhiorimetry (DSF) analysis of EBV-460 after various storage conditions and lengths. For freeze-thaws, protein was flash frozen in liquid nitrogen and immediately thawed in a 37 °C water bath for the indicated number of rounds.

[0046] FIGS. 21A-21C. Alignment of D2C3 (EBV-460) structure with two HCMV gB prefusion structures. (FIG. 21A) D2C3 (EBV-460) aligned with membrane-anchored HCMV gB construct (PDB:7KDP) (1). (FIG. 21B) D2C3 (EBV-460) aligned with the- 10 -4930-2385-3138, v. 1HCMV gB ectodomain construct (PDB: 8VYM). The black arrow highlights the movement of domain I (DI) between D2C3 (EBV-460) and the soluble HCMV gB ectodomain construct. (FIG. 21C) Pairwise alignments of DI-DIV between D2C3 (EBV-460) and the HCMV gB ectodomain construct. D2C3 (EBV-460) is shaded by domain as in FIG. 9. HCMV gB is as indicated as in (FIG. 21B). All alignments were performed using the MatchMaker function in ChimeraX. Individual domains were aligned based on residues within the respective domain.

[0047] FIGS. 22A-22B. Alignment of D2C3 (EBV-460) structure with the AlphaFold2 model of prefusion EBV gB. (FIG. 22A) D2C3 (EBV-460; orange) aligned with the EBV gB prefusion model (AlphaFold2, pink). (FIG. 22B) Pairwise alignments of DI-DIV between D2C3 (EBV-460) and the EBV gB prefusion model. D2C3 is shaded by domain as in FIG. 9. The EBV gB prefusion model is as indicated in (FIG. 22A). All alignments were performed using the MatchMaker function in ChimeraX. Individual domains were aligned based on residues within the respective domain.DETAILED DESCRIPTION

[0048] Provided herein are engineered EBV gB fusion proteins that have one or more amino acid substitutions that stabilize the EBV gB fusion protein in the prefusion conformation which can result in a substantial increase in expression and / or thermostability. Prefusion EBV gB can be used as a vaccine antigen or reagent to detect and / or isolate antibodies in sera. The prefusion EBV gB proteins described herein, and the nucleic acids that encode the proteins, may be used, for example, as potential immunogens / antigens in an immunogenic and / or antigenic composition or a vaccine against EBV, in a method of inducing an immune response in a subject, and as diagnostic tools, among other uses.I. Proteins of the Disclosure

[0049] Five glycoproteins on the viral envelope mediate EBV attachment and membrane fusion. Among these, glycoprotein B (gB) functions as the fusion protein and is therefore essential for the infection of all susceptible cell types. EBV gB is translated as a single peptide decorated by eight N-linked glycans and five O-linked glycans (Bagdonaite Journal of Biological Chemistry 2016). The gB monomer is covalently linked by five intraprotomer disulfide bonds and trimerizes to form the metastable prefusion gB conformation (Backovic Virology 2007, Backovic PNAS 2009). EBV gB also contains a- 11 -4930-2385-3138, v. 1furin cleavage site and furin cleavage enhances viral fusion (Sorem J. General Virol. 2009), although mature virions display both cleaved and uncleaved gB (Johannsen PNAS 2004). The rearrangement of gB from the metastable prefusion state to the stable postfusion state, following receptor recognition, ultimately results in fusion of the viral and host-cell membranes.

[0050] The present disclosure provides engineered proteins that include amino acid mutations relative to the amino acid sequence of the corresponding wild-type EBV gB. The amino acid mutations include amino acid substitutions, deletions, or additions relative to a wild-type EBV gB. Accordingly, the engineered proteins are mutants of wild-type EBV gBs.

[0051] The engineered proteins may possess certain beneficial characteristics, such as increased expression levels, increased stability, and / or being immunogenic and / or antigenic. The engineered proteins may possess increased immunogenic and / or antigenic properties of the prefusion conformation or improved stability in the prefusion conformation, as compared to the corresponding wild-type EBV gB. Stability refers to the degree to which a transition of the EBV gB conformation from prefusion to postfusion is hindered or prevented. The engineered proteins may display one or more introduced mutations as described herein, which may also result in improved stability in the prefusion conformation. The introduced amino acid mutations in the EBV gB include amino acid substitutions, deletions, and / or additions. The mutations in the amino acid sequences of the engineered proteins may be amino acid substitutions, insertions, and / or deletions relative to a wild-type EBV gB ectodomain. The introduced amino acid mutations may covalently link domains I and II to domain V of EBV gB.

[0052] The engineered proteins may be isolated, i.e., separated from EBV gB proteins having a postfusion conformation. Thus, the engineered proteins may be, for example, at least 80% isolated, at least 90% isolated, at least 95% isolated, at least 98% isolated, at least 99% isolated, or at least 99.9% isolated from EBV gB polypeptides in a postfusion conformation. The engineered proteins may specifically bind to an EBV gB prefusion-specific antibody.

[0053] It will be understood that a population of engineered proteins in a particular conformation can include variations (such as polypeptide modification variations, e.g., glycosylation state), that do not alter the conformational state of the engineered proteins. The population of engineered proteins may remain homogeneous over time. For example, the- 12 -4930-2385-3138, v. 1engineered proteins, when dissolved in aqueous solution, may form a population of proteins stabilized in the prefusion conformation for at least 12 hours, at least 24 hours, at least 48 hours, at least one week, at least two weeks, or more. A person of ordinary skill in the art will appreciate that the engineered proteins provided herein are useful to elicit immune responses in mammals to EBV.

[0054] The engineered ectodomains or engineered proteins may include at least one mutation selected from any one of the mutations, or sets of mutations, in Table 1. The substitutions in each of the constructs described in Table 1 were introduced into a base construct having the amino acid sequence shown in SEQ ID NO: 26 (Base). The amino acid sequence of the base construct ectodomain only (i.e., without the signal sequence or any C- terminal modifications) corresponds to amino acids 22-688 of SEQ ID NO: 26. A foldon trimerization motif of T4 fibritin (Fd) is fused to the C-terminus of the gB ectodomain by a Gly-Ser linker in the base construct. The immature construct further includes a signal sequence at the N-terminus, and an HRV3C protease recognition site, an octa-histidine tag, and a tandem Twin-Strep-tag at the C-terminus.Table 1. Exemplary Engineered Proteins with EBV gB Ectodomains (all positions relative to SEQ ID NO: 1)- 13 -4930-2385-3138, v. 14930-2385-3138, v. 1 - 14-4930-2385-3138, v. 1 - 15-4930-2385-3138, v. 1 - 16-4930-2385-3138, v. 1 - 17-4930-2385-3138, v. 1 - 18-4930-2385-3138, v. 1 - 19-4930-2385-3138, v. 1 -20-4930-2385-3138, v. 1 -21-4930-2385-3138, v. 1 -22-4930-2385-3138, v. 1 -23-4930-2385-3138, v. 1 -24-4930-2385-3138, v. 1 -25-4930-2385-3138, v. 1 -26-4930-2385-3138, v. 1 -27-4930-2385-3138, v. 1 -28-4930-2385-3138, v. 1 -29-4930-2385-3138, v. 1 -30-4930-2385-3138, v. 1 -31-4930-2385-3138, v. 1 -32-4930-2385-3138, v. 1 -33-4930-2385-3138, v. 1 -34-4930-2385-3138, v. 1 -35-4930-2385-3138, v. 1 -36-4930-2385-3138, v. 1 -37-4930-2385-3138, v. 1 -38-4930-2385-3138, v. 1 -39-4930-2385-3138, v. 1 -40-4930-2385-3138, v. 1 -41-4930-2385-3138, v. 1 -42-4930-2385-3138, v. 1 -43-Table 2. Exemplary Native EBV gB Protein Sequences

[0055] Taken together, and without being held by theory or mechanism, three requirements are suggested as being involved in maintaining gB in its prefusion conformation. First, domain I is locked near the globular core of gB. This may be achieved, for example, through the A175C / E634C substitutions in EBV gB. Second, domain V is prevented from transitioning to its extended postfusion conformation. This is achieved, for4930-2385-3138, v. 1 - 44 -example, through the I89C / L628(GCG) and A175C / E634C substitutions in EBV gB. Finally, proximity between domains II and III is maintained, and may be achieved, for example, through the H316I / D320Q / S325L substitutions.

[0056] In some embodiments, the engineered EBV gB ectodomain comprises positions 22-688 of SEQ ID NO: 1, or an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of positions 22-688 of SEQ ID NO: 1, and the amino acid substitutions W112H, Y113R, W193R, L194V, I195E, W196A, E / D220E, K / E / T241T, S / P416P, P / S423S, R428G, R429G, R430S, R431G, R432G, D / N433N, and T / A444A, where the positions are relative to SEQ ID NO: 1. The engineered EBV gB ectodomain protein may have an amino acid sequence at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of positions 22-688 of SEQ ID NO: 78.

[0057] In some embodiments, the engineered EBV gB ectodomain comprises positions 22-688 of SEQ ID NO: 1, or an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of positions 22-688 of SEQ ID NO: 1, and the amino acid substitutions W112H, Y113R, W193R, L194V, I195E, W196A, E / D220E, K / E / T241T, S / P416P, P / S423S, R428G, R429G, R430S, R431G, R432G, D / N433N, T / A444A, I89C / L628XCX (where X is any amino acid), and A175C / E634C, where the positions are relative to SEQ ID NO: 1. The engineered EBV gB ectodomain protein may have an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of positions 22-688 of SEQ ID NO: 440.

[0058] In some embodiments, the engineered EBV gB ectodomain comprises positions 22-688 of SEQ ID NO: 1, or an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of positions 22-688 of SEQ ID NO: 1, and the amino acid substitutions W112H, Y113R, W193R, L194V, I195E, W196A, E / D220E, K / E / T241T, S / P416P, P / S423S, R428G, R429G, R430S, R431G, R432G, D / N433N, T / A444A, H316VD320Q / S325L, I89C / L628XCX (where X is any amino acid), and A175C / E634C, where the positions are relative to SEQ ID NO: 1. The engineered EBV gB4930-2385-3138, v. 1 - 45 -ectodomain protein may have an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of positions 22-688 of SEQ ID NO: 485.

[0059] In some embodiments, the engineered EBV gB ectodomain includes a first cysteine substitution or insertion at any one of positions 620-640 and a second cysteine substitution or insertion at a position that introduces a non-native disulfide bond that stabilizes the recombinant EBV gB protein in the prefusion conformation, wherein the second cysteine substitution or insertion is at any one of positions 53-63, 86-91, 174, 175, 212-216, 225-227, 499-502, 517, 518, and 525-529. In some embodiments, the engineered EBV gB ectodomain includes a first cysteine substitution or insertion at any one of positions 584-587 and a second cysteine substitution or insertion at a position that introduces a non-native disulfide bond that stabilizes the recombinant EBV gB protein in the prefusion conformation, wherein the second cysteine substitution or insertion is at any one of positions 652-667. In some embodiments, the engineered EBV gB ectodomain includes a first cysteine substitution or insertion at any one of positions 648-650 and a second cysteine substitution or insertion at a position that introduces a non-native disulfide bond that stabilizes the recombinant EBV gB protein in the prefusion conformation, wherein the second cysteine substitution or insertion is at any one of positions 672-676. The Ca-Ca distance between the first cysteine substitution and the second cysteine substitution may be less than or equal to 8 A based on the three- dimensional structure of the EBV gB ectodomain prefusion conformation (Gao et al., Scientific Reports, 10:10330, 2020) (see FIG. 8).

[0060] Engineered EBV gB ectodomain designs that are described as including L628GCG or L634GCG substitutions, are also contemplated as L628XCX or L623XCX substitutions, respectively, where X can be any amino acid or absent. In hydrophobic environments, X may be, for example, alanine, leucine, isoleucine, or valine. In charged environments, X may be, for example, serine or threonine. In environments benefitting from cavity filling, X may be, for example, tyrosine, tryptophan, or phenylalanine.

[0061] Engineered EBV gB ectodomain designs that are described as comprising R428G, R429G, R430S, R431G, R432G substitutions are also contemplates as comprising any elimination of this furin cleavage site located at positions 428-432. The amino acids at these positions can each be independently substituted to another amino acid, or deleted, such that the remaining sequence is anything other than RX(K / R)R. For example, the EBV gB4930-2385-3138, v. 1 - 46 -protein ectodomain may comprise R428G, R429G, R430S, R431G, and R432G substitutions. As another example, the EBV gB protein ectodomain may comprise R429E and R430E substitutions and a deletion of one of positions R431 or R432.

[0062] The engineered protein comprising the EBV gB ectodomain may further include linkage to a trimerization domain (for example, via a Gly-Ser peptide linker). The trimerization domain may be a T4 fibritin trimerization domain (Fd) trimerization domain. The engineered protein comprising the EBV gB ectodomain protein may comprise the amino acid sequence of SEQ ID NOs: 78 or 440, or an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NOs: 78 or 440. The trimerization domain may be a a GCN4 domain; a 4J4A domain; or a two-helix bundle comprising complementary first heptad repeat (HR1) and second heptad repeat (HR2) regions of an enveloped viral fusion protein (see e.g., US Publication 2020 / 0040042, which is incorporated by reference herein in its entirety).

[0063] The engineered protein comprising the EBV gB ectodomain may further include linkage to an endosomal sorting complex required for transport (ESCRT)-recruiting domain via a transmembrane domain. The ESCRT-recruiting domain may be from EBOV VP40, Syntenin-1, rat Galectin-3 (rGalectin-3), Hrs, CD2AP, EIAV p9, HIV-1 p6, a Gag protein, and / or the ESCRT and ALIX binding region (EABR) of the human CEP55 protein. The ESCRT-recruiting domain-linked EBV gB ectodomain may be capable of being presented on the surface of a cell in which the engineered proteins are expressed. The ERD may be inserted or fused to the engineered protein N-terminally, C-terminally, or internally. Without being bound by any theory or mechanism, ESCRT-recruiting domain may recruit one or more ESCRT proteins to the cytoplasmic tail of the engineered protein. The recruitment of ESCRT proteins via the ESCRT-recruiting domain may induce the selfassembly and budding of enveloped nanoparticles (ENPs). The ESCRT-recruiting domain- linked EBV gB ectodomain may further comprise an endocytosis-preventing motif, which may tether the engineered protein to the cytoskeleton. The endocytosis-preventing motif may be from a portion of an FcR domain, such as, for example, a low-affinity gamma Fc region receptor II isoform FcRII-Bl. The endocytosis-preventing motif may comprise all or a portion of the cytoplasmic tail of FcRII-Bl. The EBV gB protein ectodomain may comprise or further comprises R837E and R838E substitutions. The EBV gB protein ectodomain may comprise4930-2385-3138, v. 1 - 47 -or further comprise an elimination of the furin cleavage site located at positions 428-432. The amino acids at these positions can each be independently substituted to another amino acid, or deleted, such that the remaining sequence is anything other than RX(K / R)R. For example, the EBV gB protein ectodomain may comprise R428G, R429G, R430S, R431G, and R432G substitutions. The engineered protein may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 22-967 of SEQ ID NO: 74.II. Protein PreparationL0064J The protein described herein may be prepared by routine methods known in the art, such as by expression in a recombinant host system using a suitable vector. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and HIGH FIVE cells (a clonal isolate derived from the parental Trichoplusia ni BTI-TN-5B1-4 cell line). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, ExpiCHO-S cells, human embryonic kidney cells (HEK293 or Expi 293 cells, typically transformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx.®. cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus-vectored systems, are known to those of skill in the art. Materials and methods for some mammalian cell and baculovirus / insect cell expression systems are commercially available in kit form. Avian cell expression systems are also known to those of skill in the art. Similarly, bacterial and mammalian cell expression systems are also known in the art.

[0065] A number of suitable vectors for expression of recombinant proteins in insect or mammalian cells are well-known and conventional in the art. Suitable vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, a terminator), and / or one or more translation signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., of mammalian origin or from a heterologous mammalian or non-mammalian species). For example, for expression in insect cells a suitable4930-2385-3138, v. 1 - 48 -baculovirus expression vector, such as PFASTBAC, is used to produce recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express recombinant protein. For expression in mammalian cells, a vector that will drive expression of the construct in the desired mammalian host cell (e.g., Chinese hamster ovary cells) is used.

[0066] The proteins can be purified using any suitable methods. For example, methods for purifying a protein by immunoaffinity chromatography are known in the art. Suitable methods for purifying desired proteins including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelating and size exclusion are known in the art. Suitable purification schemes can be created using two or more of these or other suitable methods. If desired, the protein may include a “tag” that facilitates purification, such as an epitope tag or a histidine tag. Such tagged proteins can be purified, for example from conditioned media, by chelating chromatography or affinity chromatography.III. Nucleic Acids Encoding Proteins

[0067] Also provided are nucleic acid molecules that encode a protein described herein. These nucleic acid molecules include DNA, cDNA, and RNA sequences. Nucleic acid molecules that encode only the ectodomain of the protein are also contemplated. The nucleic acid molecule can be incorporated into a vector, such as an expression vector.

[0068] The nucleic acid may be a self-replicating RNA molecule. The nucleic acid may include a modified RNA molecule. Also provided are compositions comprising a nucleic acid described herein.IV. Formulations and Methods of Use Thereof

[0069] Provided herein are compositions and methods of using the proteins described herein, or a nucleic acid encoding the proteins described herein. For example, the proteins described herein can be delivered directly as a component of an immunogenic and / or antigenic composition or a vaccine. Alternatively, nucleic acids that encode the proteins described herein can be administered to produce the protein or immunogenic and / or antigenic fragment in vivo. Protein formulations, recombinant nucleic acids (e.g., DNA, RNA, mRNA, self-replicating RNA, or any variation thereof) and / or viral vectors (e.g., live, single-round,4930-2385-3138, v. 1 - 49 -non-replicative assembled virions, or otherwise virus-like particles, or alphavirus replicon particles (VRPs)) that contain sequences encoding the proteins provided herein may be included in a composition.

[0070] Also provided are compositions including a polynucleotide that may elicit an immune response in a mammal. The polynucleotide encodes at least one polypeptide of interest, e.g., an antigen. Antigens disclosed herein may be wild type (i.e., derived from the infectious agent) or preferably modified (e.g., engineered, designed or artificial). The nucleic acid molecules described herein, specifically polynucleotides, may encode one or more engineered EBV gB protein. Such peptides or polypeptides may serve as an antigen or antigenic molecule. The term “nucleic acid’’ includes any compound that includes a polymer of nucleotides. These polymers are referred to as “polynucleotides.” Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), including mRNA, and deoxyribonucleic acids (DNAs).

[0071] The composition may include DNA encoding an engineered EBV gB protein or fragment thereof described herein. The composition may include RNA encoding an engineered EBV gB protein or fragment thereof described herein. The composition may include an mRNA polynucleotide encoding an engineered EBV gB protein or fragment thereof described herein. Such compositions may produce the appropriate protein conformation upon translation.

[0072] The composition may include at least one polynucleotide encoding two or more immunogenic and / or antigenic polypeptides or an immunogenic and / or antigenic fragment or epitope thereof. The composition may include two or more polynucleotides encoding two or more immunogenic and / or antigenic polypeptides or immunogenic and / or antigenic fragments or epitopes thereof. The one or more immunogenic and / or antigenic polypeptides may be encoded on a single polynucleotide or may be encoded individually on multiple (e.g., two or more) polynucleotides.

[0073] A composition may include (a) a polynucleotide encoding an engineered EBV gB protein; and (b) a polynucleotide encoding an additional polypeptide.

[0074] An RNA polynucleotide includes any nucleic acid sequence selected from any one of the nucleic acid sequences disclosed herein, or homologs thereof having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identity with a nucleic acid sequence disclosed herein. An4930-2385-3138, v. 1 - 50 -open reading frame may be codon-optimized. A composition may include at least one RNA polynucleotide encoding at least one EBV immunogenic and / or antigenic polypeptide or an immunogenic and / or antigenic fragment thereof and at least one 5' terminal cap. A 5' terminal cap may be 7mG(5')ppp(5')NImpNp.

[0075] The at least one polynucleotide may have at least one chemical modification. The at least one polynucleotide may further include a second chemical modification. The polynucleotide may be RNA. The at least one polynucleotide having at least one chemical modification may have a 5' terminal cap. The at least one chemical modification may be selected from pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, Nl- ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-l-methyl-l- deaza-pseudouridine, 2-thio-l -methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4- methoxy -pseudouridine, 4-thio-l -methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-0-methyl uridine. At least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracils in the open reading frame may have a chemical modification, optionally wherein the composition is formulated in a lipid nanoparticle. All of the uracils in the open reading frame may have a chemical modification. The chemical modification may be in the 5 -position of the uracil. The chemical modification may be an N1 -methyl pseudouridine.

[0076] Provided herein are methods of inducing an immune response in a mammal, the methods including administering to the mammal a composition in an amount effective to induce an immune response, wherein the composition includes a polynucleotide encoding an engineered EBV gB protein. The immune response may include a T cell response or a B cell response. The method may involve a single administration of the composition. The method may further include administering to the subject a booster dose of the composition. The composition may include a polynucleotide disclosed herein formulated in an effective amount to produce an antigen specific immune response in a mammal.

[0077] The immunogenic and / or antigenic composition may include an adjuvant. Exemplary adjuvants to enhance effectiveness of the composition include: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in- water emulsion formulations (with or without other specific adjuvants such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (PCT4930-2385-3138, v. 1 - 51 -Publ. No. WO 90 / 14837), containing 5% Squalene, 0.5% TWEEN 80, and 0.5% Span 85 formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic -blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (3) saponin adjuvants, such as QS-21, STIMULON™ (Cambridge Bioscience, Worcester, Mass.), which may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (IL-1 , IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) toll-like receptor agonists; and (7) other substances that act as adjuvants to enhance the effectiveness of the composition. The composition may not include an adjuvant. The composition may further include a lipid nanoparticle. The composition may be formulated in a nanoparticle. The composition may further include a cationic or polycationic compound, including protamine or other cationic peptides or proteins, such as poly-L-lysine (PLL).

[0078] Each of the immunogenic and / or antigenic compositions discussed herein may be used alone or in combination with one or more other antigens, the latter either from the same viral pathogen or from another pathogenic source or sources. These compositions may be used for prophylactic (to prevent infection) or therapeutic (to treat disease after infection) purposes.

[0079] The composition may include a “pharmaceutically acceptable carrier,” which includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as adjuvants. Furthermore, the antigen may be conjugated to a protein carrier, such as a ferritin, or a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and etc. pathogens. For example, a4930-2385-3138, v. 1 - 52 -disclosed engineered EBV gB ectodomain can be linked to a ferritin subunit to construct a ferritin nanoparticle. Ferritin nanoparticles and their use for immunization purposes have been disclosed in the art (see, e.g., Kanekiyo et al., Nature, 499: 102-106, 2013, incorporated by reference herein in its entirety). Ferritin is a globular protein that is found in all animals, insects, bacteria, and plants, and which acts primarily to control the rate and location of polynuclear Fe(III)2O3 formation through the transportation of hydrated iron ions and protons to and from a mineralized core.

[0080] The composition may include a diluent, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0081] The compositions described herein may include an immunologically effective amount of the polypeptide or polynucleotide, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount,” it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for eliciting an immune response. The immune response elicited may be sufficient, for example, for treatment and / or prevention and / or reduction in incidence of illness, infection or disease. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g., nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor’s assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0082] The composition may be administered parenterally, e.g., by injection, either subcutaneously, intramuscularly, or intradermally. The composition may be administered to the mammal by subcutaneous, intramuscular, or intradermal injection. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, nasal formulations, suppositories, and transdermal applications. Oral formulations may be preferred for certain viral proteins. Dosage treatment may be a single dose schedule or a multiple dose schedule. The immunogenic and / or antigenic composition may be administered in conjunction with other immunoregulatory agents.4930-2385-3138, v. 1 - 53 -

[0083] Also provided are methods of eliciting an immune response against EBV, comprising administering to a subject in need thereof an immunologically effective amount of the engineered EBV gB protein and / or an immunogenic and / or antigenic composition described herein, which comprises the proteins, DNA molecules, RNA molecules (e.g., selfreplicating RNA molecules or mRNA molecules), or VRPs as described above. The immune response may comprise the production of neutralizing antibodies against EBV.

[0084] An immune response can comprise a humoral immune response, a cell- mediated immune response, or both. An immune response may be induced against each delivered EBV protein. A cell-mediated immune response can comprise a Helper T-cell (Th) response, a CD8+ cytotoxic T-cell (CTL) response, or both. The immune response may comprise a humoral immune response comprising antibody-presenting B cells, and the antibodies may be neutralizing antibodies.

[0085] The polypeptide and / or immunogenic and / or antigenic composition described herein may also elicit an effective immune response to reduce the likelihood of an EBV infection of a non-infected mammal, or to reduce symptoms following an infection or in an already infected mammal, e.g., reduce the number of outbreaks, EBV shedding, and risk of spreading the virus to other mammals.

[0086] Provided herein are methods for reducing EBV viral shedding in a mammal. Also provided are methods for reducing EBV viral titers in a mammal. The methods may reduce EBV nucleic acids in serum in a mammal. The term “viral shedding” is used herein according to its plain ordinary meaning in medicine and virology and refers to the production and release of virus from an infected cell. The virus may be released from a cell of a mammal. Virus may be released into the environment from an infected mammal. Virus may be released from a cell within a mammal.

[0087] The methods may include administering the engineered EBV gB protein and / or immunogenic and / or antigenic composition described herein to a mammal that is infected with or is at risk of an EBV infection. The reduction in EBV viral shedding in a mammal is as compared to the viral shedding in mammals that were not administered the engineered EBV gB protein for prophylactic or therapeutic administration or those same mammals investigated prior to and post administration of the engineered EBV gB protein for therapeutic administration.4930-2385-3138, v. 1 - 54 -

[0088] The mammal may be a human. The human may be a child, such as an infant. The human may be female, including an adolescent female, a female of childbearing age, a female who is planning pregnancy, a pregnant female, and females who recently gave birth. The human may be a transplant patient or a patient awaiting a transplant.

[0089] A useful measure of antibody potency in the art is “50% neutralization titer.” Another useful measure of antibody potency is any one of the following: a “60% neutralization titer”; a “70% neutralization titer”; a “80% neutralization titer”; and a “90% neutralization titer.” To determine, for example, a 50% neutralizing titer, serum from immunized animals is diluted to assess how dilute serum can be yet retain the ability to block entry of 50% of infectious viruses into cells. For example, a titer of 700 means that serum retained the ability to neutralize 50% of infectious virus after being diluted 700-fold. Thus, higher titers indicate more potent neutralizing antibody responses. The titer may be in a range having a lower limit of about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000. The 50%, 60%, 70%, 80%, or 90% neutralization titer range can have an upper limit of about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 8000, about 9000, about 10000, about 1 1000, about 12000, about 13000, about 14000, about 15000, about 16000, about 17000, about 18000, about 19000, about 20000, about 21000, about 22000, about 23000, about 24000, about 25000, about 26000, about 27000, about 28000, about 29000, or about 30000. For example, the 50% neutralization titer can be about 3000 to about 6500. “About” means plus or minus 10% of the recited value. Neutralization titer can be measured as described in the specific examples, below.

[0090] An immune response can be stimulated by administering proteins, DNA molecules, RNA molecules (e.g., mRNA molecules, self-replicating RNA molecules or nucleoside modified RNA molecules), or VRPs to an individual, typically a mammal, including a human. The immune response induced may be a protective immune response, i.e., the response reduces the risk or severity of or clinical consequences of an EBV infection. Stimulating a protective immune response is particularly desirable in some populations particularly at risk from EBV infection and disease. For example, at-risk populations include solid organ transplant (SOT) patients, bone marrow transplant patients, and hematopoietic4930-2385-3138, v. 1 - 55 -stem cell transplant (HSCT) patients. DNA molecules, RNA molecules, or VRPs can be administered to a transplant donor pre-transplant, or a transplant recipient pre- and / or posttransplant. Because vertical transmission from mother to child is a common source of infecting infants, administering DNA molecules, RNA molecules, or VRPs to a patient who is pregnant or can become pregnant is particularly useful.

[0091] Any suitable route of administration can be used. For example, a composition can be administered intramuscularly, intraperitoneally, subcutaneously, intradermally, or transdermally. Administration may be intra-mucosal, such as intra-orally, intra-nasally, intra- vaginally, and in tra-rec tally. Compositions can be administered according to any suitable schedule.

[0092] Also provided herein is a method of inhibiting EBV entry into a cell, comprising contacting the cell with the immunogenic and / or antigenic composition described herein.

[0093] A composition may include an engineered EBV gB protein described herein. A composition may include a nucleic acid molecule or vector encoding such protein. A composition may include a protein described above and a nucleic acid molecule or vector encoding such protein.

[0094] In the case that a nucleic acid molecule encoding an engineered EBV gB protein is used in a pharmaceutical composition, the nucleic acid molecule may comprise or consist of deoxyribonucleotides and / or ribonucleotides, or analogs thereof, covalently linked together. A nucleic acid molecule as described herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g. , phosphoramidate, phosphorothioate, phosphorodithioate, or O- methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. A nucleic acid molecule may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The4930-2385-3138, v. 1 - 56 -term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the doublestranded form. A nucleic acid molecule is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “nucleic acid sequence” is the alphabetical representation of a nucleic acid molecule. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (<?.g. , degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0095] The nucleic acids of the present disclosure may comprise one or more modified nucleosides comprising a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties. In some embodiments, modified sugar moieties are substituted sugar moieties. In some embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.

[0096] In some embodiments, modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2' and / or 5' positions. Examples of sugar substituents suitable for the 2'- position, include, but are not limited to: 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'- O(CH2)2OCH3 ("MOE"). In certain embodiments, sugar substituents at the 2' position is selected from allyl, amino, azido, thio, O-allyl, O-C1-C10 alkyl, O-C1-C10 substituted alkyl; OCF3, O(CH2)2SCH3, O(CH2)2-O-N(Rm)(Rn), and 0-CH2-C(=0)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted Cl -CIO alkyl. Examples of sugar substituents at the 5'-position, include, but are not limited to: 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy. In some embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, T-F-5’-methyl sugar moieties (see, e.g., PCT4930-2385-3138, v. 1 - 57 -International Application WO 2008 / 101157, for additional 5',2’-bis substituted sugar moieties and nucleosides).

[0097] Nucleosides comprising 2'-substituted sugar moieties are referred to as 2'- substituted nucleosides. In some embodiments, a 2'-substituted nucleoside comprises a 2'- substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2-O-N(Rm)(Rn) or O-CH2- -C(=O)— N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted Cl -CIO alkyl. These 2'-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

[0098] In some embodiments, a 2'-substituted nucleoside comprises a 2'-substituent group selected from F, NH2, N3, OCF3, O-CH3, O(CH2)3NH2, CH2— CH=CH2, O- CH2— CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O-(CH2)2-O-N(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O-CH2-C(=O)-N(Rm)(Rn) where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted Cl -CIO alkyl.

[0099] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety comprising a 2’-substituent group selected from F, OCF3, O— CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2-O-N(CH3)2, -O(CH2)2O(CH2)2N(CH3)2, and O-CH2- C(=O)-N(H)CH3.

[0100] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, O-CH3, and OCH2CH2OCH3.

[0101] In some embodiments, nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present disclosure comprise one or more modified nucleobases.

[0102] In some embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted4930-2385-3138, v. 1 - 58 -purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2- amino- adenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine, 3 -deazaguanine and 3 -deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4- b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido|4,5-b|indol-2-one), pyridoindole cytidine (H-pyrido|3',2’:4,5|pyrrolo|2,3- d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Patent 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., 1991; and those disclosed by Sanghvi, Y. S., 1993.

[0103] Representative United States Patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Patents 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121 ; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of which is herein incorporated by reference in its entirety.

[0104] Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. For example, one additional modification of the ligand conjugated oligonucleotides of the present disclosure involves chemically linking to the4930-2385-3138, v. 1 - 59 -oligonucleotide one or more additional non-ligand moieties or conjugates which enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., 1989), cholic acid (Manoharan et al., 1994), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., 1992; Manoharan et al., 1993), a thiocholesterol (Oberhauser et al., 1992), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., 1991; Kabanov et al., 1990; Svinarchuk et al., 1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995; Shea et al., 1990), a polyamine or a polyethylene glycol chain (Manoharan et al., 1995), or adamantane acetic acid (Manoharan et al., 1995), a palmityl moiety (Mishra et al., 1995), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., 1996). In some aspects, a nucleic acid molecule encoding an engineered EBV gB protein is a modified RNA, such as, for example, a modified mRNA. Modified (m)RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, N1 -methylpseudouridine (Nlm ) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. In some embodiments, the (m)RNA molecules used herein may have the uracils replaced with psuedouracils such as l-methyl-3'- pseudouridylyl bases. In some embodiments, some of the uracils are replaced, but in other embodiments, all of the uracils have been replaced. The (m)RNA may comprise a 5’ cap, a 5’ UTR element, an optionally codon optimized open reading frame, a 3' UTR element, and a poly(A) sequence and / or a polyadenylation signal.

[0105] The nucleic acid molecule, whether native or modified, may be delivered as a naked nucleic acid molecule or in a delivery vehicle, such as a lipid nanoparticle. A lipid nanoparticle may comprise one or more nucleic acids present in a weight ratio to the lipid nanoparticles from about 5:1 to about 1: 100. In some embodiments, the weight ratio of nucleic acid to lipid nanoparticles is from about 5:1, 2.5: 1, 1 :1, 1:5, 1: 10, 1 :15, 1 :20, 1 :25, 1:30, 1:35, 1 :40, 1 :45, 1 :50, 1 :60, 1 :70, 1 :80, 1:90, or 1 : 100, or any value derivable therein.

[0106] In some embodiments, the lipid nanoparticles used herein may contain one, two, three, four, five, six, seven, eight, nine, or ten lipids. These lipids may include triglycerides, phospholipids, steroids or sterols, PEGylated lipids, or a group with an4930-2385-3138, v. 1 - 60 -ionizable group such as an alkyl amine and one or more hydrophobic groups such as C6 or greater alkyl groups.

[0107] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more steroid or a steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms.

[0108] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more PEGylated lipids (or PEG lipids). In some embodiments, the present disclosure comprises using any lipid to which a PEG group has been attached. In some embodiments, the PEG lipid is a diglyceride which also comprises a PEG chain attached to the glycerol group. In other embodiments, the PEG lipid is a compound which contains one or more C6-C24 long chain alkyl or alkenyl group or a C6-C24 fatty acid group attached to a linker group with a PEG chain. Some non-limiting examples of a PEG lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified l,2-diacyloxypropan-3-amines, PEG modified diacylglycerols and dialkylglycerols. In some embodiments, PEG modified diastearoylphosphatidylethanolamine or PEG modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by the molecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about 15,000. Some non-limiting examples of lipids that may be used in the present disclosure are taught by U.S. Patent 5,820,873, WO 2010 / 141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.

[0109] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more phospholipids. In some embodiments, any lipid which also comprises a4930-2385-3138, v. 1 - 61 -phosphate group. In some embodiments, the phospholipid is a structure which contains one or two long chain C6-C24 alkyl or alkenyl groups, a glycerol or a sphingosine, one or two phosphate groups, and, optionally, a small organic molecule. In some embodiments, the small organic molecule is an amino acid, a sugar, or an amino substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipid defines lipid and lipid-like molecules with both a positive charge and a negative charge.

[0110] In some aspects of the present disclosure, lipid nanoparticle containing compounds containing lipophilic and cationic components, wherein the cationic component is ionizable, are provided. In some embodiments, the cationic ionizable lipids contain one or more groups which is protonated at physiological pH but may deprotonated and has no charge at a pH above 8, 9, 10, 11, or 12. The ionizable cationic group may contain one or more protonatable amines which are able to form a cationic group at physiological pH. The cationic ionizable lipid compound may also further comprise one or more lipid components such as two or more fatty acids with C6-C24 alkyl or alkenyl carbon groups. These lipid groups may be attached through an ester linkage or may be further added through a Michael addition to a sulfur atom. In some embodiments, these compounds may be a dendrimer, a dendron, a polymer, or a combination thereof.

[0111] In some aspects of the present disclosure, composition containing compounds containing lipophilic and cationic components, wherein the cationic component is ionizable, are provided. In some embodiments, ionizable cationic lipids refer to lipid and lipid-like molecules with nitrogen atoms that can acquire charge (pKa). These lipids may be known in the literature as cationic lipids. These molecules with amino groups typically have between 2 and 6 hydrophobic chains, often alkyl or alkenyl such as C6-C24 alkyl or alkenyl groups, but may have at least 1 or more than 6 tails.

[0112] In some embodiments, the amount of the lipid nanoparticle with the nucleic acid molecule encapsulated in the pharmaceutical composition is from about 0.1% w / w to about 50% w / w, from about 0.25% w / w to about 25% w / w, from about 0.5% w / w to about 20% w / w, from about 1% w / w to about 15% w / w, from about 2% w / w to about 10% w / w, from about 2% w / w to about 5% w / w, or from about 6% w / w to about 10% w / w. In some4930-2385-3138, v. 1 - 62 -embodiments, the amount of the lipid nanoparticle with the nucleic acid molecule encapsulated in the pharmaceutical composition is from about 0.1% w / w, 0.25% w / w, 0.5% w / w, 1% w / w, 2.5% w / w, 5% w / w, 7.5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, 50% w / w, 55% w / w, 60% w / w, 65% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w, to about 95% w / w, or any range derivable therein.

[0113] In some aspects, the present disclosure comprises one or more sugars formulated into pharmaceutical compositions. In some embodiments, the sugars used herein are saccharides. These saccharides may be used to act as a lyoprotectant that protects the pharmaceutical composition from destabilization during the drying process. These water- soluble excipients include carbohydrates or saccharides such as disaccharides such as sucrose, trehalose, or lactose, a trisaccharide such as fructose, glucose, galactose comprising raffinose, polysaccharides such as starches or cellulose, or a sugar alcohol such as xylitol, sorbitol, or mannitol. In some embodiments, these excipients are solid at room temperature. Some non-limiting examples of sugar alcohols include erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotritol, maltotetraitol, or a polyglycitol.

[0114] In some embodiments, the amount of the sugar in the pharmaceutical composition is from about 25% w / w to about 98% w / w, 40% w / w to about 95% w / w, 50% w / w to about 90% w / w, 50% w / w to about 70% w / w, or from about 80% w / w to about 90% w / w. In some embodiments, the amount of the sugar in the pharmaceutical composition is from about 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, 50% w / w, 52.5% w / w, 55% w / w, 57.5% w / w, 60% w / w, 62.5% w / w, 65% w / w, 67.5% w / w, 70% w / w, 75% w / w, 80% w / w, 82.5% w / w, 85% w / w, 87.5% w / w, 90% w / w, to about 95% w / w, or any range derivable therein.

[0115] In some embodiments, the pharmaceutically acceptable polymer is a copolymer. The pharmaceutically acceptable polymer may further comprise one, two, three, four, five, or six subunits of discrete different types of polymer subunits. These polymer subunits may include polyoxypropylene, polyoxyethylene, or a similar subunit. In particular, the pharmaceutically acceptable polymer may comprise at least one hydrophobic subunit and at least one hydrophilic subunit. In particular, the copolymer may have hydrophilic subunits4930-2385-3138, v. 1 - 63 -on each side of a hydrophobic unit. The copolymer may have a hydrophilic subunit that is polyoxyethylene and a hydrophobic subunit that is polyoxypropylene.

[0116] In some embodiments, expression cassettes are employed to express an EBV gB protein, either for subsequent purification and delivery to a cell / subject, or for use directly in a viral-based delivery approach. Provided herein are expression vectors which contain one or more nucleic acids encoding an EBV gB protein.

[0117] Expression requires that appropriate signals be provided in the vectors and include various regulatory elements such as enhancers / promoters from both viral and mammalian sources that drive expression of the engineered EBV gB protein in cells. Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.

[0118] The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the EBV thymidine kinase (7A) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0119] At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl4930-2385-3138, v. 1 - 64 -transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0120] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0121] In certain embodiments, viral promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3 -phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.

[0122] Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.4930-2385-3138, v. 1 - 65 -

[0123] Below is a list of promoters / enhancers and inducible promoters / enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[0124] The promoter and / or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and / or DQ 0, 0-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, 0-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, oc-fetoprotein, t-globin, 0-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), oci-antitrypain, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus.

[0125] Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0126] There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign4930-2385-3138, v. 1 - 66 -genetic material but can be readily introduced in a variety of cell lines and laboratory animals.

[0127] One method for in vivo delivery involves the use of an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an engineered EBV gB protein that has been cloned therein. In this context, expression does not require that the gene product be synthesized.

[0128] The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

[0129] Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5’- tripartite leader (TPL) sequence which makes them preferred mRNAs for translation. In one system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral4930-2385-3138, v. 1 - 67 -vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.

[0130] Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins. Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete.

[0131] Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

[0132] The adenoviruses of the disclosure are replication defective, or at least conditionally replication defective. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is one exemplary starting material that may be used to obtain the conditional replication-defective adenovirus vector for use in the present disclosure.

[0133] Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells.

[0134] In embodiments, particular embodiments, the vector is an AAV vector. AAV is a small virus that infects humans and some other primate species. AAV is not currently4930-2385-3138, v. 1 - 68 -known to cause disease. The virus causes a very mild immune response, lending further support to its apparent lack of pathogenicity. In many cases, AAV vectors integrate into the host cell genome, which can be important for certain applications, but can also have unwanted consequences. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell, although in the native virus some integration of virally carried genes into the host genome does occur. These features make AAV a very attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. Recent human clinical trials using AAV for gene therapy in the retina have shown promise. AAV belongs to the genus Dependoparvovirus , which in turn belongs to the family Parvoviridae. The virus is a small (20 nm) replication-defective, nonenveloped virus.

[0135] The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.

[0136] With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural cap) and packaging (rep) proteins can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene. However, it was also published that the ITRs are not the only elements required in cis for the effective replication and encapsidation. A few research groups have identified a sequence designated cis-acting Rep-dependent element (CARE) inside the coding sequence of the rep gene. CARE was shown to augment the replication and encapsidation when present in cis.

[0137] In some aspects, the present disclosure provides pharmaceutical compositions that contain one or more salts. The salts may be an inorganic potassium or sodium salt such as potassium chloride, sodium chloride, potassium phosphate dibasic, potassium phosphate monobasic, sodium phosphate dibasic, or sodium phosphate monobasic. The pharmaceutical composition may comprise one or more phosphate salts such to generate a phosphate buffer solution. The phosphate buffer solution may be comprise each of the phosphates to buffer a4930-2385-3138, v. 1 - 69 -solution to a pH from about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or any range derivable therein.

[0138] In some aspects, the present disclosure comprises one or more excipients formulated into pharmaceutical compositions. An “excipient” refers to pharmaceutically acceptable carriers that are relatively inert substances used to facilitate administration or delivery of an API into a subject or used to facilitate processing of an API into drug formulations that can be used pharmaceutically for delivery to the site of action in a subject. Furthermore, these compounds may be used as diluents in order to obtain a dosage that can be readily measured or administered to a patient. Non-limiting examples of excipients include polymers, stabilizing agents, surfactants, surface modifiers, solubility enhancers, buffers, encapsulating agents, antioxidants, preservatives, nonionic wetting or clarifying agents, viscosity increasing agents, and absorption-enhancing agents.

[0139] The term “pharmaceutically acceptable” may mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and can preferably include an adjuvant. Water is a particular carrier when the pharmaceutical composition is administered by injections, such an intramuscular injection. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0140] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof,4930-2385-3138, v. 1 - 70 -preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

[0141] Engineered proteins or nucleic acids encoding engineered proteins of the present disclosure, as described herein, can be formulated for parenteral administration, e.g. , formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, intra- tumoral or even intraperitoneal routes. The formulation could alternatively be administered by a topical route directly to the mucosa, for example by nasal drops, inhalation, or by nebulizer. Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0142] Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0143] The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc. , and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0144] Dosage can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different4930-2385-3138, v. 1 - 71 -routes. Multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

[0145] The compositions disclosed herein may be used to treat both children and adults. Thus, a human subject may be less than 1 year old, 1-5 years old, 5-16 years old, 16- 55 years old, 55-65 years old, or at least 65 years old.

[0146] Preferred routes of administration include, but are not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, and intraocular injection. Particularly preferred routes of administration include intramuscular, intradermal and subcutaneous injection.V. Antibodies and Diagnostic Uses

[0147] The polypeptides described above may be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected animal or mammal (e.g., mouse, rabbit, goat, guinea pig, horse, etc.) is immunized with an immunogenic and / or antigenic polypeptide bearing an EBV epitope(s). Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an EBV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.

[0148] Monoclonal antibodies directed against EBV epitopes can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA or viruses known to immortalize B lymphocytes such as EBV. Alternatively, the general methodology for making monoclonal antibodies by single B cell sorting is known. B cells from an immunized or otherwise convalescent human or animal are sorted utilizing the EBV epitope of interest as a “bait” and epitopes that are not desirable as a negative selection. Isolated B cells are single cell sequenced and the antibody encoding genes are subcloned into an expression vector. Antibodies are subsequently expressed from the expression vector. Panels of monoclonal antibodies produced against EBV epitopes can be4930-2385-3138, v. 1 - 72 -screened for various properties; i.e., for isotype, epitope affinity, neutralizing potency against the virus, etc.

[0149] Antibodies, both monoclonal and polyclonal, which are directed against EBV epitopes are particularly useful in diagnosis, and those which are neutralizing are useful in passive immunotherapy. Monoclonal antibodies, in particular, may be used to raise antiidiotype antibodies.

[0150] Both the polypeptides which react immunologically with serum containing EBV antibodies, and the antibodies raised against these polypeptides, may be useful in immunoassays to detect the presence of EBV antibodies, or the presence of the virus, in biological samples, including for example, blood or serum samples. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. For example, the immunoassay may utilize the polypeptide having the sequence set forth in any one of SEQ ID NOs: 26-559.

[0151] Alternatively, the immunoassay may use a combination of viral antigens derived from the polypeptides described herein. It may use, for example, a monoclonal antibody directed towards at least one polypeptide described herein, a combination of monoclonal antibodies directed towards the polypeptides described herein, monoclonal antibodies directed towards different viral antigens, polyclonal antibodies directed towards the polypeptides described herein, or polyclonal antibodies directed towards different viral antigens. Protocols may be based, for example, upon competition, or direct reaction, or may be sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

[0152] Kits suitable for immunodiagnostics and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the engineered EBV gB proteins containing EBV epitopes or antibodies directed against epitopes in suitable containers, along with the remaining reagents and materials required for the conduct of the assay, as well as a suitable set of assay instructions.4930-2385-3138, v. 1 - 73 -

[0153] The polynucleotide probes can also be packaged into diagnostic kits. Diagnostic kits include the probe DNA, which may be labeled; alternatively, the probe DNA may be unlabeled and the ingredients for labeling may be included in the kit. The kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, for example, standards, as well as instructions for conducting the test.VI. Immunodetection Methods

[0154] The present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting EBV gB protein. While such methods can be applied in a traditional sense, another use will be in quality control and monitoring of vaccine stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long term stability) of antigens. Alternatively, the methods may be used to screen various antibodies for appropriate / desired reactivity profiles.

[0155] Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of EBV gB protein also is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, the immunobinding methods include obtaining a sample suspected of containing EBV gB protein, and contacting the sample with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.

[0156] These methods include methods for detecting or purifying EBV gB protein from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the EBV gB protein will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the EBV gB protein-expressing cells immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column.

[0157] The immunobinding methods also include methods for detecting and quantifying the amount of EBV gB protein or related components in a sample and the detection and quantification of any immune complexes formed during the binding process.4930-2385-3138, v. 1 - 74 -Here, one would obtain a sample suspected of containing EBV gB protein and contact the sample with an antibody that binds EBV gB protein or components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing EBV gB protein, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid (e.g., a nasal swab), including blood and serum, or a secretion, such as feces or urine.

[0158] Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to EBV gB protein. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0159] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement, as is known in the art.

[0160] The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under4930-2385-3138, v. 1 - 75 -effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0161] Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

[0162] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

[0163] Another known method of immunodetection takes advantage of the immuno- PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed out with a low pH or high salt buffer4930-2385-3138, v. 1 - 76 -that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.A. ELISAs

[0164] Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.

[0165] In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the EBV gB protein is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-EBV gB protein antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection may also be achieved by the addition of a second anti-EBV gB protein antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0166] In another exemplary ELISA, the samples suspected of containing the EBV gB protein (e.g., potentially infected cells) are immobilized onto the well surface and then contacted with the anti-EBV gB protein antibodies of the disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-EBV gB protein antibodies are detected. Where the initial anti-EBV gB protein antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-EBV gB protein antibody, with the second antibody being linked to a detectable label.

[0167] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.4930-2385-3138, v. 1 - 77 -

[0168] In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0169] In ELIS As, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

[0170] “Under conditions effective to allow immune complex (antigen / antibody) formation” means that the conditions preferably include diluting the antigens and / or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween, wherein the Tween is frequently Tween 20. These added agents also tend to assist in the reduction of nonspecific background.

[0171] The “suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25 °C to 27°C, or may be overnight at about 4°C or so.

[0172] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS / Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.4930-2385-3138, v. 1 - 78 -

[0173] To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS -Tween).

[0174] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2’-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.B. Western Blot

[0175] The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native / nondenaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

[0176] Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.

[0177] The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pl), molecular weight, electric charge, or a4930-2385-3138, v. 1 - 79 -combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

[0178] In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probing. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies.C. Immunohistochemistry

[0179] The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al. , 1990; Abbondanzo et al., 1990; Allred et al., 1990).4930-2385-3138, v. 1 - 80 -

[0180] Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and / or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and / or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings.

[0181] Permanent- sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing / pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted.D. Immunodetection Kits

[0182] In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect EBV gB protein, the antibodies may be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to an EBV gB protein, and optionally an immunodetection reagent.

[0183] In certain embodiments, the antibody may be pre-bound to a solid support, such as a column matrix and / or well of a microtitre plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[0184] Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of4930-2385-3138, v. 1 - 81 -exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.

[0185] The kits may further comprise a suitably aliquoted composition of EBV gB protein, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

[0186] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.E. Flow Cytometry and FACS

[0187] The antibodies of the present disclosure may also be used in flow cytometry or FACS. Flow cytometry is a laser- or impedance-based technology employed in many detection assays, including cell counting, cell sorting, biomarker detection and protein engineering. The technology suspends cells in a stream of fluid and passing them through an electronic detection apparatus, which allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of disorders, especially blood cancers, but has many other applications in basic research, clinical practice and clinical trials.

[0188] Fluorescence-activated cell sorting (FACS) is a specialized type of cytometry. It provides a method for sorting a heterogenous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescent characteristics of each cell. In general, the technology involves a cell suspension entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescence of each cell4930-2385-3138, v. 1 - 82 -is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based immediately prior to fluorescence intensity being measured, and the opposite charge is trapped on the droplet as it breaks form the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge.

[0189] In certain embodiments, to be used in flow cytometry or FACS, the antibodies of the present disclosure are labeled with fluorophores and then allowed to bind to the cells of interest, which are analyzed in a flow cytometer or sorted by a FACS machine.V II. Definitions

[0190] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term “portion” can include part of a moiety or the entire moiety.

[0191] As used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0192] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the disclosed subject matter. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope4930-2385-3138, v. 1 - 83 -of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0193] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0194] As used herein, a “prefusion conformation” refers to a structural conformation adopted by the polypeptide that differs from the EBV gB postfusion conformation at least in terms of molecular dimensions or three-dimensional coordinates of some or all of the molecule. The prefusion conformation refers to a structural conformation adopted by EBV gB prior to triggering of the fusogenic event that leads to transition of gB to the postfusion conformation. Isolating EBV gB in a stable prefusion conformation may be useful in informing and directing development of improved vaccines and immunogenic and / or antigenic compositions to address the important public health problem of EBV infections. A prefusion conformation may be a conformation that can bind to a prefusion-specific antibody. An engineered prefusion EBV gB may exhibit a prefusion stability that is increased at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% relative to an EBV gB sequence having at least 90% identity to wildtype EBV gB (e.g., positions 22-688 of SEQ ID NO: 1) or to EBV-53. The prefusion stability of an engineered EBV gB may be assessed by DSF or DLS, preferably DSF.

[0195] The term “antibody” refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes, for instance, chimeric, humanized, fully human, and bispecific4930-2385-3138, v. 1 - 84 -antibodies. An “antibody” is a species of an antigen binding protein. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains. Antibodies can be derived solely from a single source, or can be “chimeric,” that is, different portions of the antibody can be derived from two different antibodies as described further below. The antigen binding proteins, antibodies, or binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[0196] Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, each pair having one full-length “light” (in certain embodiments, about 25 kDa) and one full- length “heavy” chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that can be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within full-length light and heavy chains, typically, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its4930-2385-3138, v. 1 - 85 -entirety for all purposes). The variable regions of each light / heavy chain pair typically form the antigen binding site.

[0197] The term “variable region” or “variable domain” refers to a portion of the light and / or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. In certain embodiments, variable regions of different antibodies differ extensively in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines specificity of a particular antibody for its target.

[0198] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987) or Chothia et al., Nature, 342:878-883 (1989).

[0199] In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

[0200] Definitive delineation of a CDR and identification of residues comprising the binding site of an antibody may be accomplished by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex, which can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. Various methods of analysis may be employed to identify or approximate4930-2385-3138, v. 1 - 86 -the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition and the contact definition.

[0201] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g. , Chothia et al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); “AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0202] By convention, the CDR regions in the heavy chain are typically referred to as Hl , H2, and H3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus. The CDR regions in the light chain are typically referred to as LI, L2, and L3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

[0203] The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0204] The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CHI, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the4930-2385-3138, v. 1 - 87 -CH domains are at the carboxyl-terminus, with the CH3 being closest to the carboxyterminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (including IgAl and IgA2 subtypes), IgM and IgE.

[0205] A bispecific or bifunctional antibody typically is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai et al., Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0206] The term “antigen” refers to a substance capable of inducing adaptive immune responses. Specifically, an antigen is a substance which serves as a target for the receptors of an adaptive immune response. Typically, an antigen is a molecule that binds to antigenspecific receptors but cannot induce an immune response in the body by itself. Antigens are usually proteins and polysaccharides, less frequently also lipids. As used herein, antigens also include immunogens and haptens.

[0207] An “Fc” region comprises two heavy chain fragments comprising the CHI and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0208] The “Fv region” comprises the variable regions from both the heavy and light chains but lacks the constant regions.

[0209] An antibody that “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. For example, the EBV gB protein specific antibodies of the present disclosure are specific to EBV gB protein. The antibody that binds to EBV gB protein may have a dissociation constant (Kd) of = 100 nM, = 10 nM, ^1 nM, ^0.1 nM, r less, e.g., from 10“8M to 10“13M, e.g. , from 10“9M

[0210] The term “compete” when used in the context of antigen binding proteins (e.g., antibody or antigen-binding fragment thereof) that compete for the same epitope means4930-2385-3138, v. 1 - 88 -competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or antigen- binding fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand, or a reference antibody) to a common antigen (e.g., EBV gB protein or a fragment thereof). Numerous types of competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242- 253); solid phase direct biotin-avidin EIA (see, e.g. , Kirkland et al., 1986, J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually, the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65- 70%, 70-75% or 75% or more. In some instances, binding is inhibited by at least 80-85%, 85- 90%, 90-95%, 95-97%, or 97% or more.

[0211] The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. The epitope can be either linear epitope or a conformational epitope. A linear epitope is formed by a continuous sequence of amino acids from the antigen and interacts with an antibody based on their primary structure.4930-2385-3138, v. 1 - 89 -A conformational epitope, on the other hand, is composed of discontinuous sections of the antigen’s amino acid sequence and interacts with the antibody based on the 3D structure of the antigen. In general, an epitope is approximately five or six amino acids in length. Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.

[0212] The term “host cell” means a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.

[0213] The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (z.<?., an “algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48: 1073.

[0214] In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino4930-2385-3138, v. 1 - 90 -acid or nucleotide (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. A standard comparison matrix (see, Dayhoff et al. , 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) may be also used by the algorithm.

[0215] Examples of parameters that can be employed in determining percent identity for polypeptides or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J. Mol. Biol. 48:443-453.

[0216] Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 or other number of contiguous amino acids of the target polypeptide.

[0217] The term “link” as used herein refers to the association via intramolecular interaction, e.g., covalent bonds, metallic bonds, and / or ionic bonding, or inter-molecular interaction, e.g., hydrogen bond or noncovalent bonds.

[0218] The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given signal peptide that is operably linked to a polypeptide directs the secretion of the polypeptide from a cell. In the case of a promoter, a promoter that is operably linked to a coding sequence will direct the expression of the coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.4930-2385-3138, v. 1 - 91 -

[0219] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0220] The term “polynucleotide” or “nucleic acid” includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and intemucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

[0221] The terms “polypeptide” or “protein” means a macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally-occurring and non-recombinant cell; or it is produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally occurring amino acid and polymers. The terms “polypeptide” and “protein” specifically encompass EBV gB protein binding proteins, antibodies, or sequences that have deletions from, additions to, and / or substitutions of one or more amino acid of antigen-binding protein. The term “polypeptide fragment” refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein. Fragments may be about five to 500 amino acids long. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of an EBV gB protein-binding antibody, useful fragments include but are not limited to a CDR region, a variable domain of a heavy and / or light chain, a portion of an antibody chain or just its variable region including two CDRs, and the like.

[0222] The pharmaceutically acceptable carriers are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition4930-2385-3138, v. 1 - 92 -(1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically- neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0223] As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. A subject may be a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0224] The term “therapeutically effective amount” or “effective dosage” as used herein refers to the dosage or concentration of a drug effective to treat a disease or condition. For example, with regard to the use of the monoclonal antibodies or antigen-binding fragments thereof disclosed herein to treat viral infection.

[0225] “Treating” or “treatment” of a condition or infection as used herein includes preventing or alleviating a condition or infection, slowing the onset or rate of development of a condition or infection, reducing the risk of developing a condition or infection, preventing or delaying the development of symptoms associated with a condition or infection, reducing or ending symptoms associated with a condition or infection, generating a complete or partial regression of a condition or infection, mitigating a condition or infection, curing a condition or infection, or some combination thereof.4930-2385-3138, v. 1 - 93 -

[0226] “Prevention” or “preventing” includes: (1) inhibiting the onset of a disease / infection in a subject or patient which may be at risk and / or predisposed to the disease / infection but does not yet experience or display any or all of the pathology or symptomatology of the disease / infection, and / or (2) slowing the onset of the pathology or symptomatology of a disease / infection in a subject or patient which may be at risk and / or predisposed to the disease / infection but does not yet experience or display any or all of the pathology or symptomatology of the disease / infection.

[0227] As used herein, a “vector” refers to a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. A vector may also include one or more therapeutic genes and / or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like.VIII. Examples

[0228] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Materials and Methods

[0229] EBV gB proteins were produced in Expi293 or Freestyle 293 -F suspension cell cultures and purified by liquid chromatography. Briefly, Expi293 or Freestyle 293-F (Gibco, ThermoFisher Scientific) suspension cultures were grown to approximately 4 million cells / mL, then diluted to 1 million cells / mL a few hours prior to transient transfection in fresh medium. Plasmids encoding EBV gB (e.g., EBV-53, and all other variants) were mixed with polyethyleneimine, then added to suspension cell cultures. Transfected cultures were grown4930-2385-3138, v. 1 - 94 -for three to six days at 37 °C and 8% CO2 in a shaking incubator. Culture medium was harvested by centrifugation and clarified by filtration, then passed directly over Strep-Tactin XT affinity chromatography resin (IBA Lifesciences). The resin was washed with IX PBS, then eluted using the manufacturer’ s buffer, Buffer BXT. Elution fractions were analyzed by reducing and non-reducing SDS-PAGE (see FIG. 1). Fractions containing EBV gB protein were pooled, concentrated in centrifugal filters, aliquoted and flash frozen in liquid nitrogen before further use. EBV gB protein was typically 90-95% pure after affinity chromatography, as judged by SDS-PAGE. Non-reducing SDS-PAGE is a valuable assay, to confirm the protein of interest in present but also specifically for samples that contain interprotomer disulfide bonds. In such cases, EBV gB may form a trimeric structure, where three protomers are linked by the interprotomer disulfide bonds. Therefore, in cases like that non-reducing SDS-PAGE gels enable determination of trimer and if the correct interprotomer disulfide bonds are formed as expected. For quantification, an identical rectangular region of interest (ROI) was applied across all lanes, encompassing the monomer, dimer, and trimer bands. The integrated density of each lane was plotted against distance, and the area under the curve (AUC) was calculated for each band using the “magic wand” tool. Relative yield was normalized to EBV-53 (set to 1.00). Band ratios were determined by dividing individual band AUCs by the total AUC for all three bands within a lane.

[0230] In some experiments, EBV gB proteins were further purified by size exclusion chromatography (SEC) (see FIGS. 2 and 3). Frozen EBV gB aliquots were thawed and passed over a Superose 6 Increase column. The running buffer was 2 mM Tris pH 8.0, 200 mM NaCl, and 0.02% (w / v) sodium azide or 20 mM Tris pH 8.0, 200 mM NaCl, 2 mM CaCh, and 0.02% (w / v) sodium azide. One EBV gB variant in the prefusion conformation, EBV-415 (SEQ ID 442), A frozen aliquot was thawed, buffer exchanged via dialysis into 20 mM Tris pH 8.0, 50 mM NaCl, and passed over a HiTrap Q HP Anion Exchange column. This EBV Variant was eluted by a gradient ranging from 50 to 500 mM NaCl. The absorbance at 280 nm was monitored as a function of the elution volume. EBV gB protein was typically >99% pure after size exclusion chromatography, as judged by SDS-PAGE. Fractions containing EBV gB were pooled, concentrated, aliquoted and flash frozen in liquid nitrogen before further use. Size-exclusion chromatograms were plotted in Graphpad Prism. Because EBV gB exists as a trimer, the expected molecular weight and therefore elution volume by SEC enables an understanding if the variant forms exist as trimers. All desired4930-2385-3138, v. 1 - 95 -mutations, including those which do not make interprotomer disulfide bonds, should still run as trimers by SEC because they should associate as a trimer in solution.

[0231] To evaluate the effect of various substitutions on thermostability, differential scanning fluorimetry (DSF) was performed on various constructs (see FIG. 4). Purified EBV gB variants at a final concentration of 0.513 mg / mL were mixed with a final concentration of 16.5X SYPRO Orange Protein Gel Stain (ThermoFisher) in a white, opaque 96- well plate (VWR). A Roche Light Cycler 480 II was then used to measure the solutions by continuous fluorescence scanning (Zex=465 nm, Xem=580 nm). The temperature ramp rate was 4.4 °C / minute for a temperature range of 25 °C to 95 °C. Data were plotted as the negative first derivative as a function of temperature (-dF / dT). Thermostability is a valuable assay in assisting in determining if EBV gB designs exist in the prefusion conformation because the prefusion form should a different thermostable profile compared to the postfusion form. Theoretically, because prefusion is considered a meta-stable state, meaning it exists in that form prior to collapse to a more thermodynamically stable postfusion form, its melting temperature should be lower than the postfusion form. Indeed, some screened designs show altered DSF melting profiles consistent with the development of an early melting peak.

[0232] For structural studies by negative- stain electron microscopy (see FIG. 6), purified EBV gB was diluted to 0.05 mg / mL and applied to glow-discharged copper grids with formvar / carbon film. Grids were washed twice with 4 pL of 2 mM Tris pH 8.0, 200 mM NaCl, and 0.02% (w / v) sodium azide before 4 pL of a 2% methylamine tungstate solution (NanoW) was applied to the grid for 30 seconds and subsequently blotted off., then air dried and loaded into a Japan Electron Optics Laboratory NEO ARM operating in TEM mode at 200 kV. Transmission images were recorded using a OneView camera at 50,000X magnification with the pixel count binned by 2 and the CL1 condenser aperture inserted, corresponding to a calibrated pixel size of 4.3 A / pixel. Micrographs were imported into cryoSPARC for particle picking, 2D classification, and 3D reconstruction. Negative-stain imaging and the resultant 2D classifications and 3D reconstructions enable low-resolution understanding of the EBV gB molecules. The postfusion form has a very specific structure that is discernable from the prefusion form by 2D classifications. Some structures, for example EBV-415, shows 2D classifications that show clear prefusion form compared to the dominant postfusion forms seen in EBV-48 (FIG. 6).4930-2385-3138, v. 1 - 96 -

[0233] For negative-stain electron microscopy, purified EBV gB constructs were centrifuged at >20,000 x g for 7-10 min to pellet aggregated material, then diluted to 0.05 mg / mL in 20 mM Tris (pH 8.0), 200 mM NaCl, and 0.02% (w / v) sodium azide. Copper- supported carbon grids (400-mesh Formvar) were glow discharged by an EM ACE600 high vacuum sputter coater (Leica Microsystems) for 30 s at 10 mAmps. Diluted samples were then immediately applied to glow-discharged grids and stained with 0.22 pm-filtered 2% uranyl acetate (w / v). Once air-dried, grids were loaded onto a Japan Electron Optics Laboratory (JEOL) NEOARM electron microscope equipped with a OneView camera (Gatan) and operated at 200 kV with a nominal magnification of 50,000x. The Cl aperture was inserted, and micrographs were acquired in 2k x 2k mode (pixel size: 4.32 A) and saved as .dm3 files using Digital Micrograph (Gatan). All .dm3 files were converted to .mrc format using e2proc2d.py (EMAN2) and imported into CryoSPARC (Structura Biotechnology) for contrast transfer function (CTF) correction, particle picking, and 2D classification.

[0234] For structural studies by cryo-electron microscopy (see FIG. 7), purified EBV gB proteins were applied to glow-discharged grids (UltrAuFoil grids, gold film with a gold support) in a Mark IV Vitrobot. The chamber humidity was set to 100% and the chamber temperature was set to 4 °C. Grids were blotted with filter paper and plunge-frozen in liquid ethane, then loaded into a Glacios (ThermoFisher Scientific) operating at 200 kV. Exposures were collected on a Falcon 4 direct electron detector (Gatan). Motion correction, CTF estimation, particle picking, and initial 2D classification were performed in cryoSPARC Live. After collection was complete, picked particles were classified in 2D and 3D in cryoSPARC, followed by 3D refinement. Models were fitted into the Coulomb potential maps using ChimeraX, Coot, Phenix, and ISOLDE. The structure of EBV-415, for example, shows the potential prefusion EBV gB conformation. The shorter 3D map of EBV-415 closely matches previously solved prefusion structures of HCMV gB, suggesting that a population of EBV- 415 adopts a prefusion conformation. EBV-336 and EBV-402 take on shorter 3D maps as well, although the flattened shape of the maps suggests that domain I is splayed out, thereby suggesting that these constructs take-on a prefusion-like or intermediate conformation.

[0235] For antibody binding assays, to prepare antigen conjugated beads, MagPlex®- A vidin microspheres (Diasorin) designed for postfusion gB (postfusion trimer, region 18), variant EBV-415 (pre- and postfusion trimer, region 37), variant EBV-460 (prefusion trimer, region 39), irrelevant antigen (MERS-CoV S-protein 3P.GCN4, region 13) and unconjugated4930-2385-3138, v. 1 - 97 -blank beads (Region 12), were counted using a Countess S3 cell counter (Thermo Fisher) and collected via magnetic separation. Storage buffer was removed and beads were resuspended in 3 mL PBS pH 7.4 + 1% bovine serum albumin (BSA, w / v), and 5 pg antigen / 100 kDa antigen / 1 x 106beads was added. The reaction was allowed to proceed for 2 h, then beads were washed and resuspended in 1 mL Luminex wash buffer [0.1% BSA (w / v), 0.02% Tween 20 (w / v) in PBS pH 7.4]. The post-conjugation antigenicity of each antigen was validated by measuring binding to monoclonal antibodies 3A3 and 3A5 for gB- conjugated beads and JC57-14 was used for the irrelevant control MERS antigen. All monoclonal antibodies for antigenicity validation were purchased from Genscript. To assess antibody binding, serum samples from mice were diluted 75-fold, followed by an 8-point 5- fold serial dilution series in Luminex assay diluent [1% non-fat milk (w / v), 5% FBS (w / v), 0.05% Tween 20 (w / v) in PBS pH 7.4], 25 pL of diluted serum was added to 25 pL of antigen-conjugated microsphere suspension (at least 1000 microspheres / antigen). After a 30- minute incubation, microspheres were pelleted with a magnetic separator and washed three times with 300 pL of Luminex wash buffer. Microspheres were incubated with 100 pL of goat anti-mouse phycoerythrin-labelled IgG (2 pg / mL, SouthernBiotech) for 30 min. Microspheres were then pelleted with a magnetic separator, washed three times with 300 pL of Luminex wash buffer, and resuspended in 200 pL of Luminex wash buffer for analysis. Mean fluorescence intensity (MFI) results were measured using a Luminex xMAP Intelliflex. MFI values were background-corrected by subtracting signals from unconjugated blank beads. Area under the MFI curve (AUC) was calculated using GraphPad Prism 10.4.

[0236] For viral production, live EBV containing a green fluorescent protein (Akata- EBV GFP) reporter was isolated from mutant Akata BX-1 cells, which contain a recombinant EBV with GFP / Neomycin gene placed in the BXLF1 region of the viral genome. Cells were expanded at a density of 0.3-1.0 x 106cells / mL in RPML1640, 10% heat inactivated FBS (w / v), lx GlutaMAX supplement (Gibco, 35050061), 100 U / mL Penicillin-Streptromycin (R10 media) + 350 pg / mL G418 (Coming, 30-234-CI) at 37 °C, 5% CO2 to a volume of approximately 1 L. Once expanded, cells were resuspended at 4.0 x 106cells / mL in RPML 1640 + 1% heat inactivated FBS (w / v) + lx GlutaMAX supplement (R1 media) and vims was induced by treatment with 100 pg / mL goat anti-human IgG F(ab')2 (MP Biomedicals, 0855049). Cells were incubated for 4 h and subsequently diluted in R1 media to a final cell concentration of 2.0 x 106cells / mL. Cells were incubated for 5 d and then pelleted by centrifugation at 5,000 rpm for 10 min at 4 °C. Supernatant was collected, passed through a4930-2385-3138, v. 1 - 98 -0.8 pm cellulose nitrate filter (Thermo Fisher), and treated with 100 pg / mL bacitracin. Virions were then isolated by centrifugation at 21,000 x g for 90 min at 4 °C using a fixed angle rotor. Supernatant was decanted and virus was resuspended at 100-fold concentration in RPMI-1640 + 100 pg / mL bacitracin; aliquots were stored in the vapor phase of liquid N2 until use.

[0237] Virus titer and neutralization were performed based on previously described methods. Briefly, Akata-EBV-GFP virus was titrated on 4E3 and HEK293T (ATCC, CRL- 3216) target cell lines. Virus was serially 2-fold diluted in R10 media without phenol red starting from a 1:5 starting dilution and 30 pL / well of virus (or R10 as an untreated control) was added to a 96-well black wall, transparent bottom plate (Corning, 3904). 30 pL / well of R10 media was then added to each well. Then, 4.4 x 1044E3 cells / well or 2.0 x 104HEK- 293T cells / well in 60 p L was added to each well and incubated for 3 d at 37 °C, 5% CO2. Cells were analyzed by high content imaging as described below, and the dilution of virus needed to target a 20% infectivity was determined for each virus lot.

[0238] For neutralization assays, all sera were heat inactivated at 56 °C for 30 min. B cell neutralization was assessed in 4E3 cells based on previously described methods. Briefly, 4E3 cells were maintained at 37 °C, 5% CO2 in R10 media. For neutralization in 4E3 cells, 30 pL / well of sera (or controls) were prepared in a 96-well plate at a starting dilution of 1 :5 in R10 and serially 5-fold diluted in R10. Then, 30 pL / well of EBV-GFP virus was added to each well to obtain a top dilution of 1 :10, and sera were incubated with virus for Ih at 37 °C. Uninfected cells and virus-only treatments were also prepared as positive and negative controls, respectively. 4.4 x 1044E3 cells / well in 60 p L were added to virus-containing wells and incubated for 3 d at 37 °C, 5% CO2.

[0239] Epithelial cell neutralization was assessed in HEK-293T cells. HEK-293T cells were maintained in R10 media without phenol red. For neutralization, 30 pL / well of sera (or controls) were prepared in a 96-well flat bottom plate at a starting dilution of 1:5 in R10 and serially 5-fold diluted in R10. Then, 30 pL / well of EBV-GFP virus was added to each well to obtain a top dilution of 1 : 10, and sera were incubated with virus for 1 h at 37 °C. Uninfected cells and virus-only treatments were also prepared as positive and negative controls, respectively. 2.0 x 104HEK-293T cells / well in 60 p L were added to viruscontaining wells and incubated for 3 d at 37 °C, 5% CO2.4930-2385-3138, v. 1 - 99 -

[0240] For human serum depletion, healthy, EBV-seropositive adult human serum samples, with no documented linkage to an EBV-mediated pathology or disease, were purchased from BioIVT and heat-inactivated at 56 °C for 30 minutes. Streptavidin-coated magnetic beads (Pierce, 88817) were coated with biotinylated gB antigens, or left uncoated as a mock depletion, at a density of 50 pg antigen / 1 mg of beads in PBS pH 7.4 with 10% FBS (w / v) and 0.1% Tween 20 (w / v) by end-over-end mixing at 4 °C for 2 h. Coated beads were kept at 4 °C until use. To deplete antigen specific antibodies, 0.25 mg of antigen-coated beads were magnetically adhered to a 96-well plate and binding buffer removed. Beads were incubated with 50 pl of serum overnight at 4 °C with end-over-end mixing. Beads were magnetically adhered to the plate and depleted serum was removed and stored at -80 °C until use.

[0241] For antigen production and purification, postfusion gB and EBV-460 (prefusion gB) containing either a Foldon-His-Strep or a GCN4-AviTag-His were produced in Expi293F cells and purified via His-tag purification as described below. Expi293F cells were cultured in Expi293 media (Gibco) at 37 °C, 120 RPM, and 5% CO2. For transfection, cells were grown to a cell density of 3 x 106cells and transfected with plasmid DNA at 1 pg DNA / mL of cell culture using ExpiFectamine293 transfection reagent (Gibco) according to manufacturer recommendations. For constructs containing AviTag sequences, transfections were performed using a 1:1 ratio of gB:BirA plasmid in which BirA biotin ligase is expressed under control of a CMV promoter. Transfection in this manner facilitates in-cell biotinylation of the proteins produced. Cells were harvested 4-6 days post transfection by centrifugation at 7000 x g followed by filtration using a 0.22-pm filter.

[0242] Filtered supernatant was diluted with 1 Ox PBS to a final concentration of 2.5x PBS (pH 7.4) and loaded onto a HisTrap excel (Cytiva) column pre-equilibrated in 2.5x PBS pH 7.4. Proteins were eluted with 250 mM imidazole in 2.5x PBS pH 7.4. Pooled HisExcel elutions were then concentrated using Amicon Ultra centrifugal filters (Millipore Sigma, 100 kDa cutoff) to 0.5 mL. Concentrated proteins were filtered using a 0.22-pm spin filter before further purification on a Cytiva S6 10 / 300 GL (PN 29091596) or a Cytiva Superdex 200 Increase 100 / 300GL (PN 28990944) SEC column pre-equilibrated in 20 mM Tris pH 7.5, 150 mM NaCl. Protein-containing fractions were pooled, snap-frozen, and stored at -80 °C until use.4930-2385-3138, v. 1 - 100 -

[0243] Biolayer interferometry analysis was performed using an Octet R8 instrument using His IK functionalized tips. Antibodies were procured from GenScript with human Fc domains and stored in lx TBS pH 7.4. Vn and VL sequences may be found in the Supporting Information files. Antibodies and purified gB proteins were diluted to 10 pg / mL in lx Sartorius kinetics buffer [PBS pH 7.4 + 0.1% BSA (w / v), 0.02% Tween 20 (w / v), and a microbicide, Kathon] and pipetted into black-walled, black-bottom plates (200 pL / well). His IK tips were dipped into gB protein wells until an ~0.4 nm loading threshold was reached and then subsequently dipped into antibody wells to assess binding association for 120 s. Dissociation was monitored for 30 s by moving tips into a well containing buffer alone.

[0244] For mouse immunization experiments, female BALB / c mice (age 7-8 weeks) were purchased from Charles River Laboratories and acclimated for at least one week in a contract vivarium (Pacific Immunology) prior to immunization. HisTrap-purified EBV gB antigens were diluted to 0.02 mg / mL in 5% sucrose (w / v), 20 mM Tris pH 7.5, and 150 mM sodium chloride and admixed 1:1 with AddaS03 (InvivoGen). Mice were injected intramuscularly with a 50 pL dose of antigen in each hind leg, to deliver 100 pL of vaccine formulation per immunization. To obtain blood via retroorbital bleeding, a capillary tube was inserted into the medial canthus. Blood was then transferred to an Eppendorf or BD Microtainer Blood Collection Tube (BD 365967), allowed to clot at room temperature for approximately 2 h, and then spun down at 4000 rpm in a tabletop centrifuge. The serum layer was transferred to an Eppendorf tube and frozen at -80 °C. Prior to subsequent assays, serum was heat-inactivated at 56 °C for 30 min. All mouse studies were conducted in accordance with lACUC-approved protocols.Example 1

[0245] The base constructs used for the EBV gB variants contained the EBV gB ectodomain (M81 strain, residues 1-688) followed by a C-terminal T4 fibritin (foldon) trimerization motif, an HRV 3C recognition sequence, and octa-histidine tag, and a Twin- Strep tag (FIGS. 9A and 10A). Base construct zero (also referred to as: EBV-0 in Table 1) contains the full EBV gB ectodomain of B95-8 including the native signal sequence (residues 1-688). Base construct 53 (also referred to as: EBV-53) contains the full EBV gB ectodomain of M81 with the native signal sequence (residues 1-688) and further comprises the following substitutions: W112H, Y113R, W193R, L194V, I195E, W196A, E / D220E, K / E / T241T, S / P416P, P / S423S, R428G, R429G, R430S, R431G, R432G, D / N433N, T / A444A. Thus, the4930-2385-3138, v. 1 - 101 -fusion loops in EBV-53 (WY112-113and WLIW193-196) were substituted for the corresponding HSV-2 residues (HR177 l 7xand RVEA2xX 261), the furin cleavage site 1RRRRRl2x l'2) was replaced with GGSGG, and a D220E substitution present in the B95-8 strain was also included. Using these modifications, we routinely purify approximately 1.2 mg of EBV Base from 80 mL of transiently transfected FreeStyle 293 cell cultures after 6 days of culture (Table 4).

[0246] To stabilize gB in the prefusion conformation, I90C / T630C, G177C / E634C, and G57L / V223I paired substitutions were generated in EBV gB based on a sequence alignment generated using the prefusion, membrane- anchored HCMV gB structure as a template (FIGS. 9B, 10B, 17, and 19). However, these failed to improve expression yields (Table 5; FIG. 10C). Next, a disulfide scanning approach was used to assess if residues neighboring the initial target residues could more readily form stabilizing disulfide bonds. This identified several disulfides (N88C / L628C, I89C / L628C, A175C / E634C, and Q527C / E634C) capable of increasing the trimer-to-monomer band ratios observed on nonreducing SDS-PAGE (Table 5; FIG. 10C). Differential scanning fluorimetry (DSF) showed that EBV-53 had a melting temperature (Tm) of 67°C (FIG. 10D). Most variants had Tmvalues within 3°C of EBV-53, except G57L / V223I (63°C) and G57L / V223L (59°C). Notably, Q527C / E634C displayed a unique thermal profile, with a lower Tm(48°C) and an additional thermal transition at 67°C. Negative-stain electron microscopy (nsEM) revealed that the EBV Base construct as well as the single-disulfide variants exclusively adopted the postfusion conformation (FIG. 10E), displaying the characteristic crown (domain IV) and compact base (domain V) features of postfusion gB (Backovic PNAS 2009; Heldwein Science 2006). Collectively, these data demonstrated that multiple substitutions would be required to stabilize EBV gB in a prefusion conformation.

[0247] Combinatorial designs were assessed to evaluate whether pairing stabilizing disulfides could enhance trimer formation and prefusion stability. Although all variants incorporating multiple interprotomer disulfide bonds exhibited higher trimer content than their respective single variants, those containing N88C / L628C (EBV-250 and EBV-336) exhibited lower expression than those with I89C / L628C (EBV-518 and EBV-395) (Table 6; FIG. 11A). Additionally, double-dis ulfide variants containing Q527C / E634C (EBV-336 and EBV-395) improved the trimer-to-monomer ratio on non-reducing SDS-PAGE, whereas incorporating A175C / E634C (EBV-250 and EBV-518) enhanced thermostability of the first4930-2385-3138, v. 1 - 102 -thermal transition (Tmi) (Table 6; FIG. 11B). DSF analyses also revealed unique thermal transitions compared to EBV-53 for all combinatorial designs except for EBV-250.

[0248] As EBV-336 and EBV-395 showed no monomer bands on non-reducing SDS- PAGE, these samples were evaluated by nsEM. 2D class averages of both variants revealed protrusions from a globular core (FIG. 11C, white arrows), features inconsistent with the expected compact prefusion conformation and similar to features observed in the Q527C / E634C nsEM dataset (FIG. 10E, white arrow). Given that the disulfides in EBV-336 and EBV-395 were designed to covalently link domains II and IV to domain V, these protrusions were hypothesized to represent domain I, which may hinge outward at the ‘elbow’ connecting domain I to domain II (FIG. 10B, black arrows). As such, further stabilization of the variants containing the A175C / E634C disulfide bond (EBV-250 and EBV-518), which covalently links domain I to domain V, was attempted.

[0249] Disulfide bonds incorporating L628C, located six residues upstream of E634C, were hypothesized to introduce strain in the double-disulfide variants. To address this, glycine residues were introduced flanking L628C (L628(GCG)) to increase flexibility and facilitate proper disulfide formation between I89C and L628C (Table 6, FIG. 12). Nonreducing SDS-PAGE analysis revealed that incorporation of L628(GCG) in the double- disulfide variants modestly improved yield and trimer-to-monomer ratio for constructs containing the A175C / E634C substitutions (FIG. 12A, Table 6). Analysis by DSF also revealed that this modification eliminated a third thermal transition (Ton) (FIG. 12B, Table 6). However, nsEM analysis of this construct (EBV-415) revealed a subpopulation of postfusion gB (33%), congruent with the presence of a monomer molecular weight band in non-reducing SDS-PAGE (FIG. 12C). Evaluation of EBV-402 (I89C / L628(GCG), Q527C / E634C) revealed similar protrusions as seen for EBV-518 (FIG. 12C, white arrows).

[0250] To further enhance stability, ThermoMPNN and ProScan (Diekhaus PNAS 2024, Felbringer Nucleic Acids Research 2024), tools developed to predict the effects of amino acid substitutions, were used. Rational design informed by these resources identified a set of three substitutions (H316ED320Q / S325L) and a proline substitution (T292P) that were applied to improve upon the EBV-415 design. Both EBV-460 and EBV-486 exhibited 100% trimer formation by non-reducing SDS-PAGE (FIG. 12A, Table 6) and maintained similar thermostability profiles to EBV-415 (FIG. 12B). nsEM analysis further confirmed that EBV- 460 exclusively adopted the prefusion conformation, whereas EBV-486 contained particles4930-2385-3138, v. 1 - 103 -consistent with a top-down view of postfusion gB (FIG. 12C). Additional thermostability assessments demonstrated that EBV-460 retained its structural integrity after multiple freezethaw cycles and prolonged storage at both 4°C and room temperature (FIG. 20).

[0251] To further investigate antigen recognition of the gB constructs, biolayer interferometry (BLI) was used to assess antibody binding to EBV gB (FIG. 12D). Neutralizing antibodies 3A3 and 3A5, which target domains II and IV, respectively (Zhang PNAS 2022), bound EBV-53 and EBV-460 similarly. Likewise, AMM05 (Snijder Immunity 2018), a neutralizing antibody predicted to target the disordered linker region between domains II and III (Hong Front. Immunol. 2022), bound both forms of gB. In contrast, nonneutralizing antibodies AMM02, AMM03, AMM04 (Snijder Immunity 2018), and 4F10 (patent number CN1 L5925884A) exhibited notably reduced binding to EBV-460, suggesting that they recognize postfusion epitopes that become partially occluded in the prefusion conformation. While the epitope of 4H2 has not been reported, it is described as neutralizing and, like other neutralizing antibodies in the panel, did not exhibit postfusion specificity. These results further suggested that EBV -460 adopts a prefusion conformation with distinct antigenicity compared to EBV-53.

[0252] To analyze EBV-460 in greater detail and provide a high-resolution structure of prefusion EBV gB, grids of EBV-460 were frozen and a cryo-EM dataset containing 3,895 micrographs was collected (FIGs. 12E-12G). No 2D classes consistent with postfusion gB were identified via initial unbiased blob-based picking, confirming the structural homogeneity of the construct. Subsequent template-based particle picking in CryoSPARC identified ~2.2 million initial particles (Punjani et al., Nat. Methods, 2017). After iterative refinements, a final 3.1 A resolution 3D reconstruction with C3 symmetry was generated from ~160,000 particles (FIG. 16) (Sanchez-Garcia Commun. Biol. 2021). A model was built spanning EBV gB residues 43-688, although regions corresponding to residues 106-120, 185-201, 149-152, and 398-453 displayed poor map quality, suggesting intrinsic flexibility that precluded reliable modeling. To preserve consistency with previous numbering, the inserted GCG residues (L628(GCG)) were labeled 628A, 628B, and 628c, respectively.

[0253] Comparison with the full-length prefusion HCMV gB structure (Liu Science 2021) revealed that EBV-460 maintained a similar, compact prefusion conformation. A portion of domain I located near the membrane, containing the fusion loops (112, 113 and 193-196), was disordered in the EBV-460 map, likely due to the absence of the membrane-4930-2385-3138, V. 1 - 104 -proximal region in the construct, into which the fusion loops embed. In comparison to the prefusion-like, soluble HCMV gB structure, the most notable difference was the relative location of DI, which packed more tightly into the globular core of EMV-460 (FIG. 21C, black arrow), indicating better stabilization of EBV-460 compared to prefusion-like, soluble HCMV gB.

[0254] To further assess the structural basis of EBV-460 stabilization, the cryo-EM map was examined around the engineered disulfides and stabilizing substitutions. The I89C / L628(GCG) disulfide bond was only discernible at low- stringency contour thresholds, suggesting partial occupancy, structural heterogeneity, or radiation damage from the electron beam. However, biochemical validation by non-reducing SDS-PAGE and DSF confirmed that I89C / L628(GCG) is essential for trimer formation and the thermostability of prefusion gB, even in the presence of the ThermoMPNN-derived substitutions (FIG. 13). In contrast, the A175C / E634C disulfide exhibited well-defined map features, consistent with disulfide bond formation.

[0255] The map-to-model fit was also evaluated for the three ThermoMPNN- informed substitutions and each substitution was found to be well supported by the map. As expected, the H316I and S325L mutations in domain II increase hydrophobic packing interactions at the interface between domains II, III and IV. The D320Q mutation in domain II enhances intra- and interprotomer polar interactions with domain III, improving charge balance at the domain II / domain III interface. Taken together, these findings underscore the role of both engineered disulfide bonds between domains I and II to domain V and computationally predicted substitutions at the interface of domains II, III, and IV in stabilizing prefusion EBV gB.Table 3. Average yield from 40- and 80-mL mammalian cultures of EBV variants normalized to the yield of variant EBV-53 (SEQ ID 78)4930-2385-3138, v. 1 - 105 -4930-2385-3138, v. 1 - 106-4930-2385-3138, v. 1 - 107 -4930-2385-3138, v. 1 - 108-4930-2385-3138, v. 1 - 109-4930-2385-3138, v. 1 - 110-Table 4. Effects of substitutions in the EBV Base construct. All values were calculated from non-reducing gels or DSF profiles.Table 5. Characteristics of exemplary EBV gB single variants. All values were calculated from non-reducing gels or DSF profiles.4930-2385-3138, v. 1 - I ll -Table 6. Characteristics of exemplary EBV gB combination variants. All values were calculated from non-reducing gels or DSF profiles.

[0256] Mice immunized with an exemplary engineered protein of the disclosure, EBV-460, elicited sera with enhanced neutralizing activity against EBV infection of epithelial cells and B cells compared to postfusion gB. Furthermore, EBV-460 selectively depleted a higher proportion of neutralizing antibodies from human sera than did postfusion gB, indicating increased recognition of prefusion-specific epitopes.

[0257] FIG. 15 A is a schematic of mouse immunization. To assess the immunogenicity of prefusion-stabilized EBV gB variants, BALB / c mice (n= 5 / group) were immunized at days 0 and 21 with 2 pg of either postfusion gB EBV-0-1 (postfusion gB; grey), EBV-415 (mixed pre- and postfusion gB; green), or EBV-460 (prefusion gB; pink), all formulated with AS03 adjuvant. Blood samples (sera) were collected on days 21 (post-dose 1) and 35 (post-dose 2), and antibody responses were evaluated by Luminex assays.4930-2385-3138, v. 1 - 112 -

[0258] FI4. 15 shows normalized binding response presented as the ratio of binding to prefusion gB (EBV-415 or EBV-460) divided by the ratio of binding to postfusion gB (EBV- 0-1). Binding was measured by a Luminex assay. Luminex assay results suggest that immunization with constructs containing a higher proportion of prefusion gB (EBV-460 > EBV-415 > EBV-0-1) was associated with an antibody response that preferentially targeted prefusion gB over postfusion gB (FIG. 15B). This trend was evident at day 21 and became more pronounced by day 35.

[0259] To assess whether prefusion gB immunization enhances neutralizing titers relative to immunization with postfusion gB, we performed live-virus neutralization assays in HEK293T epithelial cells and Akata- 4E3 Burkitt lymphoma cells, an EBV-negative immortalized B cell line susceptible to EBV infection (Fig. 4C) (50, 51). After the first dose, neutralizing activity against EBV infection of epithelial cells was below the limit of quantification in all immunization groups. Following the second dose, sera from mice immunized with D2C3 exhibited a 2.0- and 1.8-fold higher geometric mean ICso than sera from mice immunized with G3 or postfusion gB, respectively. For B cells, neutralization was low after the first dose across all gB groups. Following the second dose, neutralizing titers increased in all groups, with sera from mice immunized with D2C3 exhibiting a 1.8-fold higher geometric mean ICso than sera from postfusion gB -immunized mice. Neutralization potency of sera from mice immunized with G3 remained comparable to that of mice immunized with postfusion gB. The data suggest a general trend toward improved neutralizing activity following prefusion gB immunization.

[0260] FIG. 15C shows neutralization by sera from immunized mice for EBV infection (by EBV-0-1 , EV-4E5, and EBV-460) of epithelial cells (left panel) and B-cells (right panel). These live-virus neutralization assays were performed using HEK293T epithelial cells and Akata-4E3 (EBV-negative) Burkitt lymphoma cells to determine whether immunization with prefusion gB elicited higher neutralizing titers than immunization with postfusion gB. After the first dose, neutralizing activity against EBV infection of epithelial cells was below the limit of quantification in all immunization groups. However, after the second dose, sera from mice immunized with EBV-460 exhibited a 2.0- and 1.8-fold higher geometric mean ICso than sera from mice immunized with EBV-415 or EBV-0-1, respectively, although this difference did not reach statistical significance. For B cells, neutralization was low after the first dose across all groups. Following the second dose,4930-2385-3138, v. 1 - 113 -neutralizing titers increased across all immunization groups, with sera from mice immunized with EBV-460 exhibiting a 1.8-fold higher geometric mean IC50 than sera from EBV-0-1- immunized mice.

[0261] Finally, depletion studies were performed using human sera to assess the antibody response resulting from natural EBV infection. FIG. 14D shows neutralization by sera from human volunteers (healthy, male donors 32-65 years of age). Sera were depleted by postfusion gB EBV-0-1, EBV-460, or not depleted (Native Serum) prior to neutralization assays. Statistical significance was determined by two-way ANOVE followed by Tukey’s HSD test in GraphPad Prism: *P < 0.05. Depletion with EBV-460 resulted in a 1.5- and 1.1- fold greater reduction in geometric mean IC50 for epithelial and B cell neutralization, respectively, compared to depletion with EBV-0-1. These findings indicate that, as observed in murine sera, neutralizing antibodies in human sera preferentially recognize prefusion gB, reinforcing its relevance as a vaccine antigen.

[0262] These results support the inclusion of prefusion-stabilized gB in EBV vaccine development.* * *

[0263] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.4930-2385-3138, v. 1 - 114 -REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.Adams et al., PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D Biological Crystallography 66, 213-221 (2010).Backovic, G. P. Leser, R. A. Lamb, R. Longnecker, T. S. Jardetzky, Characterization of EBV gB indicates properties of both class I and class II viral fusion proteins. Virology 368, 102-113 (2007).Backovic, R. Longnecker, T. S. Jardetzky, Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B. Proceedings of the National Academy of Sciences 106, 2880- 2885 (2009).Backovic, R. Longnecker, T. S. Jardetzky, Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B. Proceedings of the National Academy of Sciences 106, 2880- 2885 (2009).Backovic, T. S. Jardetzky, R. Longnecker, Hydrophobic residues that form putative fusion loops of Epstein-Barr virus glycoprotein B are critical for fusion activity. Journal of virology 81, 9596-9600 (2007).Bagdonaite et al., Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus. Journal of Biological Chemistry 291, 12014-12028 (2016).Benton, S. J. Gamblin, P. B. Rosenthal, J. J. Skehel, Structural transitions in influenza haemagglutinin at membrane fusion pH. Nature 583, 150-153 (2020).Bjomevik et al., Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science 375, 296-301 (2022).Borcherds et al., Optimal Affinity Enhancement by a Conserved Flexible Linker Controls p53 Mimicry in MdmX. Biophysical Journal 112, 2038-2042 (2017).Bu et al., Epstein-Barr virus gp42 antibodies reveal sites of vulnerability for receptor binding and fusion to B cells. Immunity 57, 559-573. e556 (2024).4930-2385-3138, v. 1 - 115 -Bu et al., Immunization with Components of the Viral Fusion Apparatus Elicits Antibodies That Neutralize Epstein-Barr Virus in B Cells and Epithelial Cells. Immunity 50, 1305-1316.el306 (2019).Burke, E. E. Heldwein, Crystal Structure of the Human Cytomegalovirus Glycoprotein B. PLOS Pathogens 11, el005227 (2015).Byrne, J. S. McLellan, Principles and practical applications of structure-based vaccine design. Current Opinion in Immunology 77, 102209 (2022).Chang et al., A prefusion-stabilized RSV F subunit vaccine elicits B cell responses with greater breadth and potency than a postfusion F vaccine. Science translational medicine 14, eade0424 (2022).Chesnokova, L. M. Hutt-Fletcher, Fusion of Epstein-Barr Virus with Epithelial Cells Can Be Triggered by av|35 in Addition to avP6 and avf)8. and Integrin Binding Triggers a Conformational Change in Glycoproteins gHgL. Journal of Virology 85, 13214- 13223 (2011).Chesnokova, M. K. Ahuja, L. M. Hutt-Fletcher, Epstein-Barr virus glycoprotein gB and gHgL can mediate fusion and entry in trans, and heat can act as a partial surrogate for gHgL and trigger a conformational change in gB. Journal of virology 88, 12193- 12201 (2014).Chesnokova, S. L. Nishimura, L. M. Hutt-Fletcher, Fusion of epithelial cells by Epstein-Barr virus proteins is triggered by binding of viral glycoproteins gHgL to integrins avp6 or avP8. Proceedings of the National Academy of Sciences 106, 20464-20469 (2009).Connolly, T. S. Jardetzky, R. Longnecker, The structural basis of herpesvirus entry. Nature Reviews Microbiology 19, 110-121 (2021).Croll, ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallographica Section D Structural Biology 74, 519- 530 (2018).Cui et al., A novel tetrameric gp3501-470 as a potential Epstein-Barr virus vaccine. Vaccine 31, 3039-3045 (2013).Cui et al., Immunization of Rabbits with Recombinant Human Cytomegalovirus Trimeric versus Monomeric gH / gL Protein Elicits Markedly Higher Titers of Antibody and Neutralization Activity. International Journal of Molecular Sciences 20, 3158 (2019).Cui et al., Rabbits immunized with Epstein-Barr virus gH / gL or gB recombinant proteins elicit higher serum virus neutralizing activity than gp350. Vaccine 34, 4050-4055 (2016).4930-2385-3138, v. 1 - 116 -Dieckhaus, M. Brocidiacono, N. Z. Randolph, B. Kuhlman, Transfer learning to leverage larger datasets for improved prediction of protein stability changes. Proceedings of the national academy of sciences 121, e2314853121 (2024).Emsley, K. Cowtan, Coot: model-building tools for molecular graphics. Acta Crystallographica Section D Biological Crystallography 60, 2126-2132 (2004).Escalante et al., A Pentavalent Epstein-Barr Virus-Like Particle Vaccine Elicits High Titers of Neutralizing Antibodies against Epstein-Barr Virus Infection in Immunized Rabbits. Vaccines 8, 169 (2020).Escalante, L. Z. Mutsvunguma, M. Muniraju, E. Rodriguez, J. G. Ogembo, Four Decades of Prophylactic EBV Vaccine Research: A Systematic Review and Historical Perspective. Frontiers in Immunology 13 (2022).Felbinger, H. V. Ribeiro-Filho, B. G. Pierce, Proscan: a structure-based proline design web server. Nucleic Acids Research 52, W280-W286 (2024).Goddard et al. , UCSF ChimeraX: Meeting modem challenges in visualization and analysis. Protein Science 27, 14-25 (2018).Goddard et al. , UCSF ChimeraX: Meeting modem challenges in visualization and analysis. Protein Science 27, 14-25 (2018).Gong, T. Ooka, T. Matsuo, E. Kieff, Epstein-Barr vims glycoprotein homologous to herpes simplex vims gB. Journal of Virology 61, 499-508 (1987).Heldwein et al., Crystal structure of glycoprotein B from herpes simplex vims 1. Science 313, 217-220 (2006).Hong et al., Glycoprotein B antibodies completely neutralize EBV infection of B Cells. Frontiers in Immunology 13, 920467 (2022).Hsieh et al., Stmcture-based design of prefusion-stabilized human metapneumovims fusion proteins. Nature communications 13, 1299 (2022).Ito et al., Stmcture of the Kaposi’s sarcoma-associated herpesvirus gB in post-fusion conformation. Journal of Virology 99, e01533-01524 (2025).Johannsen et al., Proteins of purified Epstein-Barr vims. Proceedings of the National Academy of Sciences 101, 16286-16291 (2004).Joyce et al. , Iterative stmcture-based improvement of a fusion-glycoprotein vaccine against RSV. Nature structural & molecular biology 23, 811-820 (2016).Jumper et al., Highly accurate protein stmcture prediction with AlphaFold, nature 596, 583- 589 (2021).4930-2385-3138, v. 1 - 117 -Jumper et al., Highly accurate protein structure prediction with AlphaFold, nature 596, 583- 589 (2021).Langedijk et al., Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein. Nature Communications 15, 4629 (2024).Li et al. , A novel high throughput assay to quantify Epstein-Barr virus neutralizing antibody activity against B-cell and epithelial cell infections for vaccine and therapeutic developments. Vaccine 40, 3638-3646 (2022).Li et al. , Enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing Pfs25 to IMX313 multimerization technology. Scientific Reports 6, 18848 (2016).Liu et al. , Prefusion structure of human cytomegalovirus glycoprotein B and structural basis for membrane fusion. Science Advances 7 , eabf3178 (2021).Liu et al. , Prefusion structure of human cytomegalovirus glycoprotein B and structural basis for membrane fusion. Science Advances 7, eabf3178 (2021).Malhi et al., Immunization with a self-assembling nanoparticle vaccine displaying EBV gH / gL protects humanized mice against lethal viral challenge. Cell Reports Medicine 3, 100658 (2022).Marcink et al., Intermediates in SARS-CoV-2 spike-mediated cell entry. Science Advances 8, eabo3153 (2022).Mastronarde, Automated electron microscope tomography using robust prediction Sanchez- Garcia et al., DeepEMhancer: a deep learning solution for cryo-EM volume postprocessing. Communications biology 4, 874 (2021).McCallum, D. Veesler, Computational design of prefusion-stabilized Herpesvirus gB trimers. bioRxiv 10.1101 / 2024.10.23.619923, 2024.2010.2023.619923 (2024).McLellan et al., Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus, science 342, 592-598 (2013).McShane, R. Longnecker, Cell-surface expression of a mutated Epstein-Barr virus glycoprotein B allows fusion independent of other viral proteins. Proceedings of the National Academy of Sciences 101, 17474-17479 (2004).Miranda et al., Potent neutralization of SARS-CoV-2 variants by RBD nanoparticle and prefusion-stabilized spike immunogens, npj Vaccines 9, 184 (2024).Mirdita et al., ColabFold: making protein folding accessible to all. Nature Methods 19, 679- 682 (2022).4930-2385-3138, v. 1 - 118 -Mohl, J. Chen, K. Sathiyamoorthy, T. S. Jardetzky, R. Longnecker, Structural and mechanistic insights into the tropism of Epstein-Barr virus. Molecules and cells 39, 286-291 (2016).Molesworth, C. M. Lake, C. M. Borza, S. M. Turk, L. M. Hutt-Fletcher, Epstein-Barr Virus gH Is Essential for Penetration of B Cells but Also Plays a Role in Attachment of Virus to Epithelial Cells. Journal of Virology 74, 6324-6332 (2000).Morin et al. , Collaboration gets the most out of software. eLife 2, e01456 (2013).Pallesen et al., Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proceedings of the National Academy of Sciences 114, E7348-E7357 (2017).Powell et al., A stabilized MERS-CoV spike ferritin nanoparticle vaccine elicits robust and protective neutralizing antibody responses. bioRxiv 10.1101 / 2024.07.01.601243, 2024.2007.2001.601243 (2024).Punjani, J. L. Rubinstein, D. J. Fleet, M. A. Brubaker, cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature methods 14, 290-296 (2017).Punjani, J. L. Rubinstein, D. J. Fleet, M. A. Brubaker, cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature methods 14, 290-296 (2017).Robert, P. Gouet, Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research 42, W320-W324 (2014).Robert, P. Gouet, Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research 42, W320-W324 (2014).Sanchez-Garcia et al., DeepEMhancer: a deep learning solution for cryo-EM volume postprocessing. Communications biology 4, 874 (2021).Schindelin et al., Fiji: an open-source platform for biological-image analysis. Nature Methods 9, 676-682 (2012).Shimizu, A. Tanabe-Tochikura, Y. Kuroiwa, K. Takada, Isolation of Epstein-Barr virus (EBV)-negative cell clones from the EBV-positive Burkitt's lymphoma (BL) line Akata: malignant phenotypes of BL cells are dependent on EB V. Journal of Virology 68, 6069-6073 (1994).Si et al., Different functional states of fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate. PLoS pathogens 14, el007452 (2018).Sievers, D. G. Higgins, Clustal Omega for making accurate alignments of many protein sequences. Protein Science 27, 135-145 (2018).4930-2385-3138, v. 1 - 119 -Sievers, D. G. Higgins, Clustal Omega for making accurate alignments of many protein sequences. Protein Science 27, 135-145 (2018).Snijder et al., An antibody targeting the fusion machinery neutralizes dual-tropic infection and defines a site of vulnerability on Epstein-Barr virus. Immunity 48, 799-81 1. e799 (2018).Sorem, R. Longnecker, Cleavage of Epstein-Barr virus glycoprotein B is required for full function in cell-cell fusion with both epithelial and B cells. Journal of General Virology 90, 591 -595 (2009).Sponholtz et al., Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation. Proceedings of the National Academy of Sciences 121 (2024).Sponholtz et al., Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation. Proceedings of the National Academy of Sciences 121 (2024).Stewart-Jones et al., Structure-based design of a quadrivalent fusion glycoprotein vaccine for human parainfluenza virus types 1-4. Proceedings of the National Academy of Sciences 115, 12265-12270 (2018).Sun et al., A gB nanoparticle vaccine elicits a protective neutralizing antibody response against EBV. Cell Host & Microbe 31, 1882-1897.el810 (2023).Tang et al., EMAN2: An extensible image processing suite for electron microscopy. Journal of Structural Biology 157, 38-46 (2007). van Dongen et al., Variation of Linker Length in Ratiometric Fluorescent Sensor Proteins Allows Rational Tuning of Zn(II) Affinity in the Picomolar to Femtomolar Range. Journal of the American Chemical Society 129, 3494-3495 (2007). van Rosmalen, M. Krom, M. Merkx, Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins. Biochemistry 56, 6565-6574 (2017).Vietzen et al., Ineffective control of Epstein-Barr-virus-induced autoimmunity increases the risk for multiple sclerosis. Cell 186, 5705-5718.e5713 (2023).Wamhoff et al., Enhancing antibody responses by multivalent antigen display on thymusindependent DNA origami scaffolds. Nature Communications 15 (2024).Wang et al., Importance of Neutralizing Monoclonal Antibodies Targeting Multiple Antigenic Sites on the Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein To Avoid Neutralization Escape. Journal of Virology 92, 10.1128 / jvi.02002-02017 (2018).4930-2385-3138, v. 1 - 120 -Wei et al., A bivalent EBV vaccine induces neutralizing antibodies that block B and epithelial cell infection and confer immunity in humanized mice. bioRxiv 10.1101 / 2022.01.18.476774, 2022.2001.2018.476774 (2022).White, A. E. Ward, L. Odongo, L. K. Tamm, Viral membrane fusion: a dance between proteins and lipids. Annual review of virology 10, 139-161 (2023).Wilson, J. J. Skehel, D. Wiley, Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 289, 366-373 (1981).Wong, M. T. Meehan, S. R. Burrows, D. L. Doolan, J. J. Miles, Estimating the global burden of Epstein-Barr virus-related cancers. Journal of Cancer Research and Clinical Oncology 148, 31-46 (2022).Xing et al. , Early fusion intermediate of ACE2-using coronavirus spike acting as an antiviral target. Cell 188, 1297-1314.el224 (2025).Yin, X. Wen, R. G. Paterson, R. A. Lamb, T. S. Jardetzky, Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation. Nature 439, 38-44 (2006).Zhang et al., Protective anti-gB neutralizing antibodies targeting two vulnerable sites for EBV-cell membrane fusion. Proceedings of the National Academy of Sciences 119 (2022).4930-2385-3138, v. 1 - 121 -

Claims

WHAT IS CLAIMED IS:

1. An engineered protein comprising an engineered Epstein-Barr virus (EBV) gB protein ectodomain comprising a sequence having at least 90% identity to amino acids 22-672 of SEQ ID NO: 1, said engineered ectodomain comprising at least one set of substitutions selected from the group consisting of: Q527C / E634C, S652C / A672C, N88C / L628C, A175C / E634C, T498C / F655C, N88C / L628GCG, I89C / L628GCG, N88C / L628XCX, I89C / L628XCX, H316FD320Q / S325L, and Q460C / I461C, wherein the positions are relative to SEQ ID NO: 1.

2. An engineered protein comprising an engineered Epstein-Barr virus (EBV) gB protein ectodomain comprising a sequence having at least 90% identity to amino acids 22-672 of SEQ ID NO: 1, said engineered ectodomain comprising one or more of the following substitutions, sets of substitutions, deletions, or insertions, wherein the positions are relative to SEQ ID NO: 1 :(a) A175C / a cysteine substitution or insertion at any one of positions 620-640; A175C / E634C; A175C / E634CG; A175C / E634GC; A175C / E634GCG; A175C / E634XCX; A175C / I633C; A175C / N635C; A195W; A357P; A457F; A457N; A457P; A457W; A457Y; A464C / Y465C; A464W; A464Y; A513P; A620C / T624C; A620P; A639P; A639R; A651C / A672C; A651P; D / E220E; D133P; D320Q; D360P; D433N; D637P; D648C / A672C; D656P; D87C / L628C; D87C / N629C; D87C / S627C; D87C / a cysteine substitution or insertion at any one of positions 620-640; D87R; D87P; E195I; E356P; E398P; E496C / L497C; E634C; E642P; E648P; E658P; E663P; E665P; E79P; F463P; F46Y; F46Y / F48Y / H603Y; F48P; F48Y; F60C / F625C; F625C / S627C; F625P; F638P; F655P; F661P; F666P; F71P; G172C / D564C; G172P; G177C / E634C; G177C / I633C; G177C / N635C; G177C / a cysteine substitution or insertion at any one of positions 620-640; G227C / Q593C; G227C / T591C; G322C / A482C; G333P; G511P; G520C / F596C; G520C / Y594C; G57C / I633C; G57L; G659H; G659N; G659P; G659Q; G659W; G659Y; H112W; H316I; H316FD320Q / S325L; H56C / G227C; H56C / K214C; H56C / Q593C; H56Y; H603Y; I102D; I138P; I195E; I369P; I461C / A464C; I461C / F463C; I461F; I461P; I472P; I500C / D656C; I500C / E658C; I500C / G659C; I500C / L657C; I500D; I509P; I619F; I619Y; I626P; I633P; I636H; I636P; I660P; I89C; I89C / L628C; I89C / L628GC; I89C / L628GCG; I89C / L628XCX; I89C / N629C; I89C / S631C; I89C / T630C; I89C / a cysteine substitution or insertion at any one of positions 620-640; I89CI; I89CI / L628C; I89CVL628GC;4930-2385-3138, v. 1 - 122 -I89CFL628GCG; I89CFL628XCX; I89CFN629C; I89CVS631C; I89CVT63OC; I89CI7a cysteine substitution or insertion at any one of positions 620-640; I90C; I90C / L632C; I90C / S631C; I90C / T630C; I90C / T630GC; I90C / T630GCG; I90C / T630XCX; I90C / a cysteine substitution or insertion at any one of positions 620-640; I90V; K124R; K124Y; K124Y / T132VA639R; K214C / I633C; K241T; K354P; K540P; K499C / D656C;K499C / E658C; K499C / F655C; K499C / L657C; K499Q; K512P; K663P; K86C / I626C; K86C / L628C; K86C / S627C; L174C / I636C; L194V; L396P; L399P; L453P; L494C / R495C; L497C / T498C; L621C / L628C; L622C / I626C; L622C / L628C; L622C / N629C;L622C / S627C; L622C / a cysteine substitution or insertion at any one of positions 620-640; L622P; L628P; L632P; L633P; L641P; L657P; M135P; M212C / I633C; M492C / S508C; M506P; M584C / E663C; M584C / F666C; M584C / N665C; M584C / R662C; N176C / E634C; N176C / I633C; N176C / N635C; N454P; N455P; N473P; N491C / Q623C; N501C / E658C; N501C / G659C; N501C / L657C; N629D; N629P; N653P; N665P; N670D; N670W; N670Y; N88C / L628C; N88C / L628CG; N88C / L628GC; N88C / L628GCG; N88C / L628XCX; N88C / N629C; N88C / T630C; N88C / a cysteine substitution or insertion at any one of positions 620-640; N88NC / L628C; N88NC / L628CG; N88NC / L628GC; N88NC / L628GCG; N88NC / L628XCX; N88NC / N629C; N88NC / T630C; N88NC / a cysteine substitution or insertion at any one of positions 620-640; P423S; P502C / E658C; P502C / G659C; P502C / L657C; P91A; P91C; P91C / I633C; P91C / L632C; P91C / S631C; P91C / T630C; P91C / a cysteine substitution or insertion at any one of positions 620-640; Q134P; Q140P; Q228C / Q593C; Q298C / S627C; Q359P; Q460C / I461C; Q460P; Q462P; Q471P; Q527C / E634GCG; Q527C / E634XCX; Q527C / E634C; Q527C / L632C; Q589P;Q623C / F625C; Q623C / I626C; Q623C / L628C; Q623C / S627C; Q623P; Q649C / A672C; Q649P; Q67P; R113Y; R120I; R179C / E634C; R180G; R193W; R428X; R428G; R429X; R429G; R430X; R430S; R431X; R431G; R431S; R432X; R432G; R469P; R470P; R495C / E496C; R518I, R650C / A672C; R650C / K676C; R650C / R675C; R650P; R662P; R74C / V358C; S116C / G689C; S129W; S285I; S325C / A480C; S325L; S416P; S507P; S508P; S526C / D637C; S54C / S526C; S55C / D637C; S55C / G227C; S627P; S631D; S631P; S63C / T621C; S63P; S640I; S640P; S645T; S652C / A672C; S652P; S70C / H316C; S93T; S98I; T118C / G689C; T132I; T132L; T132P; T137P; T209E; T209I; T225C / N635C; T229C / Q593C; T292P; T397P; T400P; T401P; T403P; T444A; T452P; T458E; T458P; T498C / D656C; T498C / F655C; T498C / K499C; T498C / L657C; T585C / F666C;T585C / N665C; T585C / Q667C; T621C / T624C; T621P; T624C / I626C; T624C / S627C; T624P; T630C; T630P; V178C / E634C; V178C / I636C; V178S; V194L; V223I; V223L;4930-2385-3138, v. 1 - 123 -V230C / Q593C; V318C / A480C; V318C / G477C; V358P; V459C / I461C; V459P;V493C / L494C; V505P; V514P; V522C / Y595C; V525F; V525I; V525L; V525W;V587C / N653C; V654F; V654P; V654W; V654Y; W112H; W193R; W196A;W196C / G716C; W300C / I626C; W300C / S627C; Y113R; Y139P; Y198C / S712C; Y355P;Y465C / L468C; Y465C / S467C; Y510P; Y594R; Y664P; and / or Y92H;(b) a first cysteine substitution or insertion at any one of positions 620-640 and a second cysteine substitution or insertion at a position that introduces a non-native disulfide bond that stabilizes the recombinant EBV gB protein in the prefusion conformation, wherein the second cysteine substitution or insertion is at any one of positions 53-63, 86-91, 174, 175, 212-216, 225-227, 499-502, 517, 518, and 525-529; a first cysteine substitution or insertion at any one of positions 584-587 and a second cysteine substitution or insertion at a position that introduces a non-native disulfide bond that stabilizes the recombinant EBV gB protein in the prefusion conformation, wherein the second cysteine substitution or insertion is at any one of positions 652-667; and / or a first cysteine substitution or insertion at any one of positions 648- 650 and a second cysteine substitution or insertion at a position that introduces a non-native disulfide bond that stabilizes the recombinant EBV gB protein in the prefusion conformation, wherein the second cysteine substitution or insertion is at any one of positions 672-676;(c) replacement of positions 623-647 with PPFIDLNITMLCDHEFVPLEVYTRH; replacement of position 457 with EIRELEARIRELEIRI; replacement of positions 1-29 with MRGGGLICALVVGALVAAVASA; replacement of positions 87-231 with ENIAPYKFKATMYYKDVTVSQVWFGNRTSQFMGIFEDRAPVPFEEVIDKINAKGVCR STAKYVRNNMETTAFHRDDHETDMELKPAKVATRCSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVY; replacement of positions 170-181 with PDDYSNCHSTRY; replacement of positions 401-455 withGDEFTRNYNYLARERYLKRLLAEEGAD; replacement of positions 401-455 withGLSAAQLRQNRDAILQALAALLD; replacement of positions 401-455 withGRPPTRENLVRTRLELLRAAGAP; replacement of positions 401-455 withG VD YRDMTIEQRAELLRRTFGLD ; replacement of positions 401-455 withGVSYRDAIAAAYTPAHWEAAGVD; replacement of positions 401-455 withGVTAAERPAWRTAVLQYLAATVA; replacement of positions 401-455 withG VTP AERP AW VEA ALKA A A ATGD ; replacement of positions 422-460 withGNATTPLKQIVLRIMETEARTAK; replacement of positions 441 -460 with4930-2385-3138, v. 1 - 124 -LKQIVLRIMEIEARIAK; and / or replacement of positions 456-457 with EIRELEARIRELEIRI;(d) deletion of position 673-688; deletion of positions 403-407; deletion of positions A681- Q688; deletion of positions G685-Q688; deletion of positions N684-Q688; deletion of positions N687-Q688; deletion of positions Q688; deletion of positions R686-Q688; and / or deletion of positions S683-Q688; deletion of positions V682-Q688; and / or(e) insertion ofAPAAPAAPRASGGVAATVAANGGPASRPPPVPSPATTKARKRKTKKPPKRPEATP after position 29; or insertion ofAPAAPRASGGVAATVAANGGPASRPPPVPSPATTKARKRKTKKPPKRPEATP after position 29.

3. The engineered protein of claim 1 or 2, wherein the EBV gB protein ectodomain comprises the Q527C / E634C substitutions.

4. The engineered protein of claim 3, wherein the EBV gB protein ectodomain further comprises one or more substitution or set of substitutions selected from the group consisting of: H316I, D320Q, S325L, I89C / L628GCG, I89C / L628XCX, I89CI / L628GCG, I89CPL628XCX, K124Y, A639R, N88C / L628C, N88C / L628GCG, N88C / L628XCX, N88NC / L628C, N88NC / L628GCG, and N88NC / L628XCX.

5. The engineered protein of claim 3, wherein the EBV gB protein ectodomain further comprises H316I, D320Q, and S325L substitutions.

6. The engineered protein of claim 3, wherein the EBV gB protein ectodomain further comprises I89C / L628GCG or I89C / L628XCX substitutions.

7. The engineered protein of claim 1 or 2, wherein the EBV gB protein ectodomain comprises the S652C / A672C substitutions.

8. The engineered protein of claim 7, wherein the EBV gB protein ectodomain further comprises N88C / L628C and / or A175C / E634C substitutions.

9. The engineered protein of claim 1 or 2, wherein the EBV gB protein ectodomain comprises the N88C / L628C substitutions.4930-2385-3138, v. 1 - 125 -10. The engineered protein of claim 9, wherein the EBV gB protein ectodomain further comprises S652C / A672C and / or Q527C / E634C substitutions.

11. The engineered protein of claim 1 or 2, wherein the EBV gB protein ectodomain comprises the A175C / E634C substitutions.

12. The engineered protein of claim 1 1, wherein the EBV gB protein ectodomain further comprises S652C / A672C substitutions.

13. The engineered protein of claim 1 or 2, wherein the EBV gB protein ectodomain comprises the N88C / L628GCG or N88C / L628XCX substitutions.

14. The engineered protein of claim 13, wherein the EBV gB protein ectodomain further comprises Q527C / E634C substitutions.

15. The engineered protein of claim 1 or 2, wherein the EBV gB protein ectodomain comprises the I89C / L628GCG or I89C / L628XCX substitutions.

16. The engineered protein of claim 15, wherein the EBV gB protein ectodomain further comprises one or more substitution or set of substitutions selected from the group consisting of: H316I, D320Q, S325L, K124Y, A639R, Q527C / E634C, and A175C / E634C.

17. The engineered protein of claim 15, wherein the EBV gB protein ectodomain further comprises H316I, D320Q, and S325L substitutions.

18. The engineered protein of claim 17, wherein the EBV gB protein ectodomain further comprises Q527C / E634C substitutions.

19. The engineered protein of claim 1 or 2, wherein amino acids 401-455 of the EBV gB protein ectodomain are replaced with the amino acid sequence GVSYRDAIAAAYTPAHWEAAGVD.

20. The engineered protein of claim 19, wherein the EBV gB protein ectodomain further comprises Q527C / E634C substitutions.

21. The engineered protein of claim 1 or 2, wherein the EBV gB protein ectodomain comprises one of the following sets of substitutions:I89C / L628XCX, A175C / E634C;4930-2385-3138, v. 1 - 126 -I89C / L628GCG, A175C / E634C;I89C / L628TCG, A175C / E634C;S652C / A672C, N88C / L628C;S652C / A672C, A175C / E634C;N88C / L628C, Q527C / E634C;N88C / L628XCX, Q527C / E634C;Q527C / E634C, H316I / D32OQ / S325L;I89C / L628XCX, Q527C / E634C;D320Q, Q527C / E634C;K124Y, Q257C / E634C;I89C / L628XCX, H316UD320Q / S325L, Q527C / E634C;I89C / L628XCX, K124Y, D320Q, Q527C / E634C;I89C / L628XCX, K124Y, D320Q, A639R, Q527C / E634C;I89C / L628XCX, K124Y, H316FD320Q / S325L, Q527C / E634C;H316UD320Q / S325L;H316I / D320Q / S325L, A175C / E634C;I89C / L628XCX, H316UD320Q / S325L, A175C / E634C;I89C / L628GCG, H316UD320Q / S325L, A175C / E634C;I89C / L628XCX, H316UD320Q / S325L, A175C / E634C, T292P;I89C / L628GCG, H316UD320Q / S325L, A175C / E634C, T292P;I89C / L628XCX, H316I / D320Q / S325L, A175C / E634C, T292P, K124Y; or I89C / L628GCG, H316FD320Q / S325L, A175C / E634C, T292P, K124Y.

22. The engineered protein of any one of claims 1-21, wherein the EBV gB protein ectodomain further comprises one or more substitution selected from the group consisting of: A620P, D87P, E665P, E79P, F48P, F638P, F71P, G172P, G333P, H56Y, I369P, I633P, K540P, L622P, N665P, Q589P, Q623P, Q67P, R518I, S129W, S285I, S631P, S63P, S640I, S98I, T132L, T209I, T292P, and Y594R.

23. The engineered protein of any one of claims 1-21, wherein the EBV gB protein ectodomain further comprises one or more substitution sets selected from the group consisting of: G520C / F596C, G520C / Y594C, G57C / I633C, H56C / G227C, M492C / S508C, S325C / A480C, S526C / D637C, S54C / S526C, S55C / D637C, S55C / G227C, S63C / T621C, S70C / H316C, V318C / A480C, V 18C / G477C, and V522C / Y595C.4930-2385-3138, v. 1 - 127 -24. The engineered protein of claim 1 or 2, wherein the EBV gB protein ectodomain comprises one of the sets of substitutions, insertions, and / or deletions of Table 1.

25. The engineered protein of any one of claims 1-24, wherein the EBV gB protein ectodomain further comprises one of the following sets of substitutions:(a) W112H, Y113R, W193R, L194V, I195E, W196A, E / D220E, K / E / T241T, S / P416P, P / S423S, R428X, R429X, R430X, R431X, R432X, D / N433N, T / A444A;(b) W193R, L194V, I195E, W196A, E / D220E, K / E / T241T, S / P416P, P / S423S, R428X, R429X, R430X, R431X, R432X, D / N433N, T / A444A;(c) L194V, I195E, W196A, E / D220E, K / E / T241T, S / P416P, P / S423S, R428X, R429X, R430X, R431X, R432X, D / N433N, T / A444A;(d) W193R, T195E, W196A, E / D220E, K / E / T241 T, S / P416P, P / S423S, R428X, R429X, R430X, R431X, R432X, D / N433N, T / A444A; or(e) I195E, W196A, E / D220E, K / E / T241T, S / P416P, P / S423S, R428X, R429X, R430X, R431X, R432X, D / N433N, T / A444A.

26. The engineered protein of any one of claims 1-25, wherein the EBV gB protein ectodomain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 22-672 of SEQ ID NO: 78.

27. The engineered protein of any one of claims 1-25, wherein the EBV gB protein ectodomain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 22-672 of one of SEQ ID NOs: 26-73, 75-79, 82-160, 164- 256, 260, 273, 275, 277-322, 328-333, 338-384, 386-403, 408-453, 456-504, 506-533, and 537-559.

28. The engineered protein of any one of claims 1-27, wherein the engineered protein does not comprise the cytoplasmic tail of EBV gB.

29. The engineered protein of any one of claims 1-28, wherein the engineered EBV gB protein ectodomain is fused or conjugated to a trimerization domain.

30. The engineered protein of claim 29, wherein the engineered EBV gB protein ectodomain is fused to a trimerization domain.4930-2385-3138, v. 1 - 128 -31. The engineered protein of claim 29 or 30, wherein the dimerization domain comprises a T4 fibritin trimerization domain; a GCN4 domain; a 4J4A domain; a two-helix bundle comprising complementary first heptad repeat (HR1) and second heptad repeat (HR2) regions; or a combination thereof.

32. The engineered protein of claim 30 or 31, wherein the trimerization domain is fused to the C -terminus of the ectodomain via a linker.

33. The engineered protein of any one of claims 1-32, wherein the engineered EBV gB protein ectodomain is fused or conjugated to a transmembrane domain.

34. The engineered protein of claim 33, wherein the engineered EBV gB protein ectodomain is fused to a membrane anchor and / or transmembrane domain.

35. The engineered protein of claim 33 or 34, wherein the transmembrane domain comprises an EBV gB protein transmembrane domain.

36. The engineered protein of claim 33 or 34, wherein the transmembrane domain does not comprise an EBV gB protein transmembrane domain.

37. The engineered protein of any one of claims 1-36, which comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 22-753 of SEQ ID NO: 1.

38. The engineered protein of any one of claims 34-36, wherein the engineered protein further comprises an EBV gB cytoplasmic domain.

39. The engineered protein of claim 38, which comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 22-857 of SEQ ID NO: 1.

40. The engineered protein of claim 38 or 39, wherein the engineered protein further comprises an endosomal sorting complex required for transport (ESCRT) recruiting domain.

41. The engineered protein of claim 40, wherein the ESCRT recruiting domain comprises the sequenceALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPYGGGSFNSSINN IHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAAHSLP.4930-2385-3138, v. 1 - 129 -42. The engineered protein of claim 40 or 41, wherein the EBV gB protein cytoplasmic domain comprises R837E and R838E substitutions.

43. The engineered protein of any one of claims 40-42, which comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acids 22-967 of SEQ ID NO: 74.

44. The engineered protein of any one of claims 1-43, wherein the EBV gB protein ectodomain comprises an elimination of the furin cleavage site.

45. The engineered protein of any one of claims 1-44, wherein the EBV gB protein ectodomain further comprises one of the sets of substitutions, insertions, and / or deletions of Table 1.

46. The engineered protein of any one of claims 40-45, which comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 74, 161-163, 257-259, 274, 276, 323-327, 334-337, 385, 404-407, 454, and 455.

47. The engineered protein of any one of claims 1-46, comprising an N-terminal signal sequence.

48. The engineered protein of any one of claims 1-47, which comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1.

49. An engineered EBV gB protein trimer comprising three engineered proteins according to any one of claims 1-48.

50. A nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence of the engineered protein of any one of claims 1-48.

51. The nucleic acid molecule of claim 50, wherein the nucleic acid molecule further comprises a DNA expression vector.4930-2385-3138, v. 1 - 130 -52. The nucleic acid molecule of claim 50, wherein the nucleic acid molecule is an mRNA.

53. The nucleic acid molecule of claim 50, wherein the nucleic acid molecule is a selfreplicated RNA molecule.

54. The nucleic acid molecule of any one of claims 50-53, which further comprises at least one chemical modification.

55. The nucleic acid molecule of claim 54, wherein the at least one chemical modification is selected from the group consisting of pseudouridine, N1 -methylpseudouridine, Nl- ethylpseudouridine, Nl-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouri dine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4- methoxy -pseudouridine, 4-thio- 1 -methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyl uridine.

56. A pharmaceutical composition comprising (i) the engineered protein of any one of claims 1-48, (ii) the engineered trimer of 49, or (iii) the nucleic acid molecule of any one of claims 50-55; and a pharmaceutically acceptable carrier.

57. The pharmaceutical composition of claim 56, further comprising an adjuvant.

58. The pharmaceutical composition of claim 56 or 57, wherein the composition is formulated within a cationic lipid nanoparticle.

59. A method of treating or preventing Epstein-Barr virus (EBV) infection or a disease associated with EBV infection in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 56-58.

60. A method of eliciting an immune response in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 56-58.4930-2385-3138, v. 1 - 131 -61. A method for reducing EBV viral shedding in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 56-58.

62. The pharmaceutical composition of any one of claims 56-58 for use in the treatment or prevention of a EBV infection or a disease associated with EBV infection in a subject.

63. The method of any one of claims 59-61 or the pharmaceutical composition for use of claim 62, wherein the subject is a mammal, optionally wherein the subject is a human.

64. The pharmaceutical composition of any one of claims 56-58 for use in eliciting an immune response against EBV.

65. Use of (i) the engineered protein of any one of claims 1-48, (ii) the engineered trimer of 49, or (iii) the nucleic acid molecule of any one of claims 50-55 in the manufacture of a medicament for the treatment or prevention of an EBV infection or a disease associated with EBV infection.

66. A composition comprising (i) the engineered protein of any one of claims 1-48 or (ii) the engineered trimer of claim 49 bound to an antibody.

67. The composition of claim 66, wherein the antibody specifically binds to an EBV gB in the prefusion conformation.4930-2385-3138, v. 1 - 132 -

Images