Variants of epstein-BARR virus glycoprotein b, nucleic acids encoding the same, and uses thereof

Abstract

This application relates to variants of Epstein-Barr virus glycoprotein B and nucleic acids, such as messenger ribonucleic acids (mRNAs), encoding the same, as well as compositions comprising the same, vaccines comprising the same, and methods of using the same, such as in the prevention and / or treatment of diseases or conditions caused by Epstein-Barr virus.

Description

VARIANTS OF EPSTEIN-BARR VIRUS GLYCOPROTEIN B, NUCLEIC ACIDS ENCODING THE SAME, AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and relies on the filing date of, European Application No. 24306919.2, filed 15 November 2024, and European Application No.24306987.9, filed 27 November 2024, the entire disclosures of which are herein incorporated by reference.FIELD

[0002] This application relates to variants of Epstein-Barr virus glycoprotein B and nucleic acids, such as messenger ribonucleic acids (mRNAs), encoding the same, as well as compositions comprising the same, vaccines comprising the same, and methods of using the same, such as in the prevention and / or treatment of diseases or conditions caused by Epstein-Barr virus.BACKGROUND

[0003] Epstein Barr virus (EBV) is a ubiquitous human herpesvirus, which infects B cells of the immune system and epithelial cells and establishes lifelong latency in the host (Jean-Pierre et al., Frontiers Microbiol., 2021, 12:701611). During primary infection, EBV is associated with infectious mononucleosis (IM), and a wide spectrum of long-term associated diseases has been described, such as auto-immune diseases (e.g., multiple sclerosis) and certain cancers (e.g., Burkitt and Hodgkin lymphomas) (Bjomevik et al., Science, 2022, 375(6578):296-301; Brady et al., Postgrad. Med. J., 2008, 84(993):372-377). The risk of developing IM, the clinically significant form of primary infection with EBV, is higher if infection occurs in late childhood or adolescence. There is currently no licensed EBV vaccine for any indication on the market, but several candidates are in clinical and pre-clinical development (Escalante et al., Front. Immunol., 2022, 13:867918).

[0004] For instance, a recombinant protein, adjuvanted vaccine candidate composed of the glycoprotein gp350 of EBV and adjuvant AS04 was demonstrated in a Phase 2 efficacy study to be 78% effective at preventing IM in healthy young adults, but it was ineffective at preventing EBV infection (Sokal et al., J. Infect. Dis., 2007, 196(12): 1749-1753). This vaccine candidateinduced neutralizing antibodies preventing B cell entry but was not successful at blocking entry into epithelial cells, which might explain its failure to prevent primary EB V infection. Other EB V vaccines are also being developed, mainly in pre-clinical or in early clinical development (Rozman et al., Pathogens, 2022, 11:864).

[0005] Accordingly, there is an urgent need to develop effective EBV vaccines.SUMMARY

[0006] The present disclosure is based, in part, on the surprising discovery that introducing at least two cysteine substitutions in certain regions of the EBV gB ectodomain stabilizes the EBV gB in the prefusion conformation. Specifically, based on a combination of several approaches, including computational design and a structural based mutational approach, the inventors have found at least three regions in the EBV gB ectodomain that are important for stabilizing the EBV gB trimer in the prefusion conformation via formation of disulfide bridges.

[0007] By identifying these specific regions in the EBV gB ectodomain, the inventors have discovered a strategy for stabilizing EBV gB in the prefusion conformation that requires only minimal alterations to the EBV gB ectodomain. As such, this design strategy not only yields modified EBV gB with a stabilized prefusion conformation but also preserves naturally occurring EBV gB antigen structure and neutralizing epitopes, and is, thus, advantageous for developing EBV gB antigens (and nucleic acids encoding the same) and vaccines comprising the same.

[0008] Accordingly, in one aspect, this disclosure is directed to a variant of an Epstein-Barr virus (EBV) glycoprotein B (gB), wherein the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, and wherein the at least two cysteine substitutions are located in two regions of the EBV gB selected from a first region located at amino acid residues 49-56 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, a second region located at amino acid residues 525-529 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, and a third region located at amino acid residues 631-637 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the modified EBV gB ectodomain shares at least 95% sequence identity to the ectodomain of the EBV gB.

[0009] In certain embodiments, the cysteine substitution located in the first region of the EBV gB comprises S55C, L53C, or S54C, the cysteine substitution located in the second region of theEBV gB comprises Q527C, V525C, S526C, or V529C, and the cysteine substitution located in the third region of the EBV gB comprises L632C, N635C, I633C, E634C, or D637C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Specific pairs of cysteine substitutions falling within these regions are disclosed herein.

[0010] In certain embodiments, the variant further comprises at least two additional cysteine substitutions relative to the EBV gB, and wherein one of the at least two additional cysteine substitutions is located in a fourth region located at amino acid residues 170-177 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 and another one of the at least two additional cysteine substitutions is located in a fifth region located at amino acid residues 560-567 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, as disclosed herein.

[0011] In certain embodiments, the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, wherein the at least two cysteine substitutions are selected from Q527C, L632C, S55C, N635C, G172C, D564C, L53C, S54C, R61C, S63C, I90C, L174C, A175C, G177C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, V529C, N563C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, I633C, E634C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, and R675C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, wherein the at least two cysteine substitutions form a disulfide bridge in the modified EBV gB ectodomain, and wherein, if the variant comprises cysteine substitutions G172C and D564C, A175C and V529C, or G322C and D478C, the variant further comprises at least two additional cysteine substitutions relative to the EBV gB. In certain embodiments, the modified EBV gB ectodomain shares at least 95% sequence identity to the ectodomain of the EBV gB.

[0012] In certain embodiments, the at least two cysteine substitutions comprise Q527C and L632C, S55C and N635C, G172C and D564C, L53C and D637C, S54C and S526C, R61C and T621C, S63C and T621C, I90C and T630C, L174C and N563C, A175C and V529C, A175C and E634C, G177C and E634C, K214C and I633C, T225C and N635C, G227C and T591C, T229C and N606C, V318C and G477C, D320C and D478C, G322C and D478C, G322C and A480C, G322C and A482C, T323C and A482C, G376C and T624C, S389C and F463C, I500C and I660C, N501C and F661C, K517C and L657C, G520C and Y594C, V525C and L632C, S526C andN635C, Q527C and E634C, L580C and Y644C, T581C and Y644C, T585C and Q669C, E586C and A668C, G659C and Y664C, or R675C and A651C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0013] In certain embodiments, the variant may further comprises one or more additional substitutions in the gB ectodomain, as disclosed herein.

[0014] In another aspect, the disclosure provides a trimeric EBV gB complex, comprising three copies of any of the variants of the EBV gB as disclosed herein (i.e., three copies of the same EBV gB variant).

[0015] In another aspect, the disclosure provides an artificial nucleic acid (e.g., mRNA) encoding any of the variant EBV gB as disclosed herein, a vector comprising the artificial nucleic acid, or a host cell comprising the vector.

[0016] In another aspect, the disclosure provides an Epstein-Barr virus-like particle (VLP) comprising any of the variant EBV gB as disclosed herein, which may optionally further comprising one or more additional EBV glycoproteins selected from gH / gL, gp42, gp350, gp220, or a combination thereof, and / or T cell antigens selected from BZLF1, BMRF1, EBNA3a, EBNA3b, EBNA3c, LMP2, BRFL1, BMLF1, and / or EBNA-1.

[0017] In another aspect, the disclosure provides a composition (e.g., immunogenic composition) comprising any of the variant EBV gB as disclosed herein, a trimeric EBV gB complex as disclosed herein, an artificial nucleic acid as disclosed herein, a vector as disclosed herein, or an Epstein-Barr VLP as disclosed herein.

[0018] In another aspect, the disclosure provides an artificial messenger ribonucleic acid (mRNA) encoding a variant of an Epstein-Barr virus (EBV) glycoprotein B (gB), wherein the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, and wherein the at least two cysteine substitutions are located in two regions of the EBV gB selected from a first region located at amino acid residues 49-56 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, a second region located at amino acid residues 525-529 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, and a third region located at amino acid residues 631-637 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the modified EBV gB ectodomain encoded by artificial mRNA shares at least 95% sequence identity to the ectodomain of the EBV gB.

[0019] In certain embodiments, the cysteine substitution located in the first region of the EBV gB comprises S55C, L53C, or S54C, the cysteine substitution located in the second region of the EBV gB comprises Q527C, V525C, S526C, or V529C, and the cysteine substitution located in the third region of the EBV gB comprises L632C, N635C, I633C, E634C, or D637C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Specific pairs of cysteine substitutions falling within these regions are disclosed herein. In some embodiments, the variant of the EBV gB encoded by the artificial mRNA of the present disclosure further comprises at least two cysteine substitutions comprise Y198C and S712C and / or I771C and H802C. In some embodiments, the variant of the EBV gB encoded by the artificial mRNA of the present disclosure further comprises substitutions R837T and R838T, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0020] In certain embodiments, artificial mRNA comprise a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57 (or a nucleic acid sequence sharing at least 90% identity with any of these SEQ ID NOs).

[0021] In another aspect, the disclosure provides a composition (e.g. immunogenic composition) comprising the artificial mRNA as disclosed herein encapsulated in a lipid nanoparticle (LNP), as described herein.

[0022] In another aspect, the disclosure provides a vaccine comprising the compositions described herein, and a pharmaceutically acceptable carrier, optionally further comprising an adjuvant.

[0023] In another aspect, the disclosure provides a method of immunizing a subject (e.g., a human), the method comprising administering to the subject in need thereof the vaccine as described herein. In certain embodiments, the method prevents an Epstein-Barr virus infection in the subject, decreases the subject’s likelihood of getting an Epstein-Barr virus infection, or reduces the subject’s likelihood of getting serious illness from an Epstein-Barr virus infection.

[0024] In another aspect, the disclosure provides a method of reducing one or more symptoms of an Epstein-Barr virus infection, the method comprising administering to a subject in need thereof the vaccine as described herein.

[0025] In another aspect, the disclosure provides an in vitro method of preparing a trimeric EB V gB complex, the method comprising culturing a host cell, as described herein, in a cell culture medium, and expressing the EBV gB complex, and optionally purifying the trimeric EBV gB complex from the cell culture medium.BRIEF DESCRIPTION OF THE DRAWING

[0026] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods and compositions disclosed herein.

[0027] FIG. 1 depicts a schematic structure of a trimeric Epstein Barr virus (EBV) gB ectodomain (source: Backovic et al., PNAS, 2009, 106(8):2880-2885). The five domains of the EBV gB are indicated with roman numbers I to V. N: N terminus; C: C terminus.

[0028] FIG. 2A-2C depict representative 2-dimensional (2D) class averages from negative staining electron microscopy (nsEM) of EBV gB trimers formed with the variants of the EBV gB.FIG. 2A: construct rEBVgB_G685_S55C-N635C (Design ID # 7); FIG. 2B: construct rEBVgB_G685_Q527C-L632C (Design ID # 10); FIG. 2C: construct rEBVgB_G685_Q527C-E634C (Design ID # 11).

[0029] FIG. 3A-3C depict representative 2D class averages from nsEM of EBV gB trimers formed with the variants of the EBV gB. FIG. 3A: construct rEBVgB_G685_K517C-L657C (design ID # 25); FIG. 3B: construct rEBVgB_G685_L53C-D637C (design ID # 26); FIG. 3C: construct rEBVgB_G685_T581-Y644C (design ID # 28).

[0030] FIG. 4A-4B depict representative 2D class averages from nsEM of EBV gB trimers formed with the variants of the EBV gB. FIG. 4A: construct rEBVgB_G685_S54C-S526C_S55C-N635C_Q527C-L632C (design ID # 7-6); FIG. 4B: construct rEBVgB_G685_G172C-D564C_S55C-N635C_Q527C-L632C (design ID # 7-15).

[0031] FIG. 4C depicts representative 2D class averages from cryo-electron microscopy (cryo-EM) of EBV gB trimers from construct rEBVgB_G685_G172C-D564C_S55C-N635C_Q527C-L632C (design ID # 7-15).

[0032] FIG. 4D depicts a schematic cryo-EM structure of EBV gB trimer from construct rEBVgB_G685_G172C-D564C_S55C-N635C_Q527C-L632C (design ID # 7-15). Side view (left) and top-down view (right) of cryo-EM maps shown here resemble prefusion conformation predicted by AlphaFold 2. The relative position of domain I (DI) and domain II (DII) to central helix is highlighted.

[0033] FIG. 5A-5B depict the corrected median fluorescence intensity (MFI) detected in cells expressing the mRNA designs described in Example 4. FIG.5A: MFI detected in cells expressing the mRNA designs in small scale; FIG. 5B: MFI detected in cells expressing the mRNA designs in large scale.DETAILED DESCRIPTION

[0034] Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings and discussed in the detailed description that follows. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the disclosure, and should not be interpreted as limiting the scope of the disclosure.

[0035] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.Definitions

[0036] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0037] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merelyas labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0038] The term “about,” or “approximately,” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amount and the like, “about” is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value as such variations are appropriate to perform the disclosed methods and / or to make and use the disclosed compositions. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

[0039] An “amino acid” according to the present disclosure can be any of the twenty naturally occurring (or “standard” amino acids) or variants thereof, such as, for example, D-proline (the D-enantiomer of proline), or any variants that are not naturally found in proteins, such as norleucine. The standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties, such as cysteine that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that forms a cycle to the polypeptide backbone, and glycine that is more flexible than other amino acids. Table 1 shows the abbreviations and properties of the standard amino acids.Table 1. Standard amino acids, abbreviations, and properties.

[0040] The term “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0041] As used herein, the term “antibody” refers to an immunoglobulin molecule produced by B lymphoid cells with a specific amino acid sequence. In some embodiments, antibodies are evoked in humans or other animals by a specific antigen (immunogen). Antibodies are characterized by reacting specifically with the antigen in some demonstrable way, antibody and antigen each being defined in terms of the other. The terms “eliciting an antibody response,” “eliciting neutralizing antibody,” “eliciting immunogenic response,” or grammatical equivalents, refer to the ability of an antigen or other molecule to induce the production of antibodies. In some embodiments, the term “antibodies” refers to any recombinant antibodies used in in vitro assays,including one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Such antibodies may exist as intact immunoglobulins or as fragments of the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Exemplary antibody fragments include, but are not limited to, F(ab)'2, Fab', and single chain Fv (scFv).

[0042] As used herein, the term “antigen” refers to an agent that elicits an immune response; and / or (ii) an agent that is bound by a T cell receptor (e.g., when presented by an MHC molecule) or to a membrane-bound B cell receptor or a soluble antibody (e.g., produced by a B cell) when exposed or administered to an organism. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies) in an organism; alternatively or additionally, in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen) in an organism. It will be appreciated by those skilled in the art that a particular antigen may elicit an immune response in one or several members of a target organism (e.g., mice, ferrets, rabbits, primates, humans), but not in all members of the target organism species. In some embodiments, an antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, including all values and subranges therebetween, of the members of a target organism species. In some embodiments, an antigen binds to an antibody and / or T cell receptor and may or may not induce a particular physiological response in an organism. In some embodiments, for example, an antigen may bind to an antibody and / or to a T cell receptor in vitro, whether or not such an interaction occurs in vivo. In some embodiments, an antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. Antigens include the variants of the Epstein-Barr virus (EBV) glycoprotein B (gB) described herein.

[0043] As used herein, an “artificial nucleic acid molecule” or the like may typically be understood to be a nucleic acid, e.g., a DNA or an RNA, that does not occur naturally. Thus, an “artificial messenger ribonucleic acid (mRNA)” refers to an mRNA that does not occur naturally. In other words, an artificial nucleic acid molecule may be understood as a non-natural nucleic acid molecule or mRNA molecule. Such nucleic acid or mRNA molecule may be non-natural due to its individual sequence (which does not occur naturally) and / or due to other modifications, e.g., structural modifications of nucleotides that do not occur naturally. An artificial nucleic acid molecule may be a DNA molecule, an RNA molecule (e.g., mRNA), or a hybrid moleculecomprising DNA and RNA portions. Typically, artificial nucleic acid molecules may be designed and / or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence). Further, the term “artificial nucleic acid molecule” or the like (e.g., “artificial mRNA”) is not restricted to mean “one single molecule” but is, typically, understood to comprise an ensemble of identical molecules. Accordingly, it may relate to a plurality of identical molecules contained in an aliquot.

[0044] The phrase “as indexed by reference to the amino acid sequence of SEQ ID NO: 1,” as used herein, refers to a normalized biological sequence alignment that allows the comparison of a query sequence (e.g., the sequence of a variant of the EBV gB to which one or more of the mutations (e.g., amino acid substitutions) described herein have been or will be applied) to a subject sequence (e.g., a wild-type EBV gB sequence, such as the amino acid sequence under GenBank accession number P03188.1 as set forth in SEQ ID NO: 1), thereby identifying amino acid residues in the target sequence that correspond to the same positions in the subject sequence. In general, the target sequence and the query sequence share characteristic portions or features but differ slightly in length and / or sequence identity. For example, the numbering of residues in a specific target sequence or for targeted modification can be identified and described based on the amino acid sequence of the wild-type EBV gB polypeptide (i.e., SEQ ID NO: 1). Sequences are aligned to the full-length wild-type EBV gB polypeptide sequence, including signal peptide, transmembrane domain, and cytoplasmic tail domain) (i.e., SEQ ID NO: 1). The N-terminal methionine of the signal peptide is residue 1. Accordingly, the phrase “amino acid position x as indexed by reference to the amino acid sequence of SEQ ID NO: 1” is used herein to designate the position / identity of an amino acid residue in a polypeptide of interest (e.g., a variant of the EBV gB) by referring to the corresponding amino acid at position x in the wild-type EBV gB polypeptide sequence (i.e., SEQ ID NO: 1). Similarly, when referring to SEQ ID NO: 2, the phrase “as indexed by reference to the amino acid sequence of SEQ ID NO: 2” is used herein to designate the position / identity of an amino acid residue in a polypeptide of interest (e.g., a variant of the EBV gB) by referring to the corresponding amino acid at position x in the wild-type EBV gB polypeptide sequence (i.e., SEQ ID NO: 2).

[0045] The term “at least,” “less than,” “more than,” or “up to” prior to a number or series of numbers (e.g., “at least two”) is understood to include the number adjacent to the term “at least,” “less than” or “more than,” and all subsequent numbers or integers that could logically be included,as clear from context. When the term “at least,” “less than,” “more than,” or “up to” is present before a series of numbers or a range, it is understood that “at least,” “less than,” “more than,” or “up to” can modify each of the numbers in the series or range.

[0046] The term “biological activity,” as used herein, refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest. In some embodiments, the biological activity of an EBV gB polypeptide refers to the ability of the EBV gB polypeptide to elicit neutralizing antibodies. In these cases, the term “biological activity” is used interchangeably with “immunogenic activity.”

[0047] As used herein, a “codon-optimized” nucleic acid sequence refers to a nucleic acid sequence that has been altered such that expression of the encoded protein is improved and optimized for a particular expression system. A “codon-optimized” nucleic acid sequence encodes the same protein as a non-optimized parental sequence upon which the “codon-optimized” nucleic acid sequence is based. For example, a nucleic acid sequence may be “codon-optimized” for expression in mammalian cells (e.g., CHO cells, human cells, mouse cells etc.), bacterial cells (e.g., E. coif), insect cells, yeast cells or plant cells.

[0048] An “ectodomain,” as used herein, is the domain of a membrane protein that extends into the extracellular space (the space outside a cell). The ectodomain of an EBV gB is generally defined as the N-terminal segment located at amino acids 23-685 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. This N terminal segment corresponds to amino acid residues 23-685 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. The amino acid residue numbering is the same between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.

[0049] As used herein, the term “epitope” includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component in whole or in part. In some embodiments, an epitope is comprised of a plurality of amino acid residues in an antigen. In some embodiments, the amino acid residues are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, the amino acid residues are physicallynear to or continuous with each other in space when the antigen adopts such a conformation. In some embodiments, at least some of the amino acids are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized; e.g., a non-linear epitope).

[0050] The term “host” is used herein to refer to a system (e.g., a cell, organism, etc.) in which a polypeptide of interest is present. In some embodiments, a host is a system that is susceptible to infection with a particular infectious agent. In some embodiments, a host is a system that expresses a particular polypeptide of interest.

[0051] As used herein, the term “host cell” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. For example, host cells may be used to produce the variants of the EBV gB described herein by standard recombinant techniques. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell: as used herein. In some embodiments, host cells include any prokaryotic and eukaryotic cells suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence). Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, microalgae (including both eukaryotic algae, such as Chlamydomonas, Chlorella, Nannochlor opsis, Thraustochytriales (e.g., Schizochytrium sp.), diatoms (e.g., Phaeodactylum), and prokaryotic cyanobacteria, also known as blue-green algae such as Arthrospira), insect cells (e.g., SF-9, SF- 21, baculovirus-infected insect cells, Trichoplusiani, etc.), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO KI, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2 / 0, NS-0, MMT 060562, Sertoli cell, BRL 3 A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from anaforementioned cell. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

[0052] As used herein, the term “in some embodiments,” “in certain embodiments,” “in other embodiments,” “in some other embodiments,” or the like, refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0053] As used here, an “mRNA vaccine” refers to a type of vaccine that uses messenger RNA (mRNA) to produce an immune response.

[0054] The term “mutation” refers to deletion, addition, or substitution of an amino acid residue in the amino acid sequence of a modified protein or polypeptide as compared to the amino acid sequence of a reference protein or polypeptide.

[0055] As used herein, the term “N-linked glycosylation site” refers to a location on a polypeptide, such as a protein, where a glycan attaches to an asparagine (N) residue. An N-linked glycosylation site comprises a consensus sequence of NxS / Ty (SEQ ID NO: 17), in which N is asparagine, x and y are any residue except proline (P), and S / T is a serine or threonine residue. A glycan is a polysaccharide or oligosaccharide. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan.

[0056] The term “prevent,” “preventing,” or “prevention,” as used herein, refers to prophylaxis, avoidance of disease manifestation, a delay of onset, and / or reduction in frequency and / or severity of one or more symptoms of a particular disease, disorder or condition (e.g., infection with, for example, a virus, such as EBV). In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and / or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition.

[0057] As used herein, the term “prophylactically effective amount” means an amount sufficient to avoid disease manifestation, delay onset of and / or reduce in frequency and / or severity one or more symptoms of a particular disease, disorder or condition (e.g., infection with, for example, a virus, such as EBV).

[0058] The term “sequence identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. “Sequence identity” can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., Siam J. Applied Math., 48:1073 (1988). Typical methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity and similarity are codified in publicly available computer programs. Typical computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894: Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity. In some embodiments, the sequence identity is determined using the BLAST program with the default parameters.

[0059] As used herein, a “recombinant vaccine” refers to a type of vaccine that uses genetic engineering to produce antigens from a pathogen (e.g., EBV) using a harmless organism, such as yeast or bacteria.

[0060] As used herein, the term “subject” means any member of the animal kingdom. Typically, “subject” refers to humans. In some embodiments, “subject” refers to non-human animals. In some embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig). In some embodiments, a subject may be a transgenic animal, genetically-engineered animal, and / or a clone. In some embodiments, the subject is an adult, an adolescent or an infant. In some embodiments, the term “individual” or “patient” is used and is intended to be interchangeable with the term “subject.”

[0061] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0062] As used herein, the phrase “virus-like particle” or “VLP” refers to particles that resemble a virus yet lack any viral genetic material and, therefore, are not infectious. A “viruslike particle” or “VLP” may be produced by heterologous expression in a variety of cell culture systems including mammalian cell lines, insect cell lines, yeast, and plant cells. In addition, VLPs can be purified by methods known in the art. In some embodiments, an EBV VLP as described herein comprises the engineered EBV gB polypeptide described herein. In some embodiments, an EBV VLP as described herein comprises, in addition to the engineered EBV gB polypeptide described herein, other EBV glycoproteins, including, but are not limited to, gH / gL, gp42, gp350, gp220, BZLF1, BMRF1, EBNA3a, EBNA3b, EBNA3c, LMP2, BRFL1, BMLF1, EBNA-1, or any combination thereof.

[0063] As used herein, the term “wild-type” generally refers to a normal form of a protein or nucleic acid, as is found in nature. For example, wild-type EBV gB polypeptides are found in natural isolates of EBV. An exemplary wild-type EBV gB polypeptide sequence can be found under GenBank accession number P03188, as set forth in SEQ ID NO: 1. Another exemplary wild-type EBV gB polypeptide sequence can be found under GenBank accession number QCF57191.1, as set forth in SEQ ID NO: 2.Structure of Epstein-Barr Virus Glycoprotein B

[0064] Epstein-Barr virus (EBV) is a member of the Gammaherpesviridae subfamily of herpesviruses. EBV infects epithelial and B cells in the host, with fusion of the virus envelope with cell membranes of the host cell being a requisite step in the entry process, as with otherherpesviruses. This process requires the cooperative function of multiple viral glycoproteins. For B cells, glycoprotein 42 (gp42), the glycoprotein complex gH / gL, and glycoprotein B (gB) are essential for EBV glycoprotein-mediated fusion, whereas with epithelial cells, only gB and the gH / gL complex are essential for EBV glycoprotein-mediated fusion (Connolly et al., Nat. Rev. Microbiol., 2011, 9:369-381).

[0065] The EBV gB is an 857-amino-acid protein with a long amino-terminal ectodomain that includes nine potential N-linked glycosylation sites and a predicted 22-amino-acid cleavable signal peptide at the N terminus. The most C-terminal of three hydrophobic domains found in the primary amino acid sequence of the EBV gB is the transmembrane domain (TM), which is required for membrane anchoring (Pellett et al., J. Virol., 1985, 56:807-813; Gong et al., J. Virol., 1987, 61:499-508). Following the TM is a 104-amino-acid C-terminal cytoplasmic tail, generally referred to as the cytoplasmic tail domain (CTD) (Pellett et al., J. Virol., 1985, 56:807-813). The amino acid sequence of the full-length EBV gB precursor can be found in, for instance, the GenBank database under accession number P03188.1 and is reproduced below (signal peptide: bold and italic TM: bold; CTD: italic)'.M77? / ? / ?ELAf LZAAZAC / ?£G'AQTPEQPAPPATTVQPTATRQQTSFPFRV CELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKI VTNILIYNGWYADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGWLIWTYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPSAARGSTPAAVLRRRRRDAGNATTPVPPTAPGKSLGT LNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKIN PTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETM CYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNE IHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRASNV FDLEGIFREYNFQAQNIAGLRKDLDNAVSNGRNQFVDGLGELMDSLGS VGQSITNLVSTVGGLFSSLVSGFISFFKNPFGGMLILVLVAGVVILVISL TRRTRQMSQQPVQMLYPGIDELAQQHASGEGPGINPISKTELQAIMLALHEQNQEQKRAAQRAAGPSVASRALQAARDRFPGLRRRRYHDPETAAALLGEAETEF (SEQ ID NO: 1).

[0066] The EBV gB ectodomain, generally referred to the N-terminal segment located at amino acid residues 23-685 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, is an elongated rod-like molecule, composed of 5 domains that contain both P-sheet and a-helical secondary structures (Backovic et al., Proc. Natl. Acad. Sci. U S A, 2009, 106(8):2880-2885). Domain I (residues 89-294 as indexed by reference to the amino acid sequence of SEQ ID NO: 1) contains the gB fusion loops (FLs) and its core region has a fold that resembles that of a plekstrin-homology (PH) domain, Domain II (composed of residues 77-88 and 295-390 as indexed by reference to the amino acid sequence of SEQ ID NO: 1) has a PH domain fold, Domain III (composed of residues 52-68, 455-527, and 617-624 as indexed by reference to the amino acid sequence of SEQ ID NO: 1) features a 42-residue long aC helix, which wraps around the helices from other 2 subunits in a left-handed twist, Domain IV is made of residues 528-616 and a short N-terminal region consisting of residues 42-51 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, and Domain V (residues 625-679 as indexed by reference to the amino acid sequence of SEQ ID NO: 1) reaches across and inserts into the cavity formed by other 2 subunits, tying together the gB trimer (Backovic et al., Proc. Natl. Acad. Sci. U S A, 2009, 106(8):2880-2885). The domain structures found in the EBV gB, the fusion protein G of vesicular stomatitis virus (VSV G), and recently in the baculovirus fusion protein gp64, led to their classification as a class III type of viral fusion protein.

[0067] Like other viral fusion proteins, three monomeric EBV gB proteins wrap around each other by interacting through multiple contact surfaces to form a spike-like trimer. A schematic structure of a trimeric EBV gB ectodomain is shown in FIG. 1.

[0068] Also like other viral fusion proteins, the EBV gB trimer tends to change from a high-energy, metastable prefusion state to a highly stable postfusion state, which is irreversible (Si et al., PloS Pathog., 2018, 14(12):el007452; Zeev-Ben-Mordehai et al., Proc. Natl. Acad. Sci. U S A, 2016, 113(15):4176-4181). The transition from prefusion to postfusion involves a major structural change in the EBV gB protein, allowing it to effectively facilitate membrane fusion. Thus, in the context of the EBV, “prefusion” EBV gB refers to the initial, folded state of the viral gB protein before it triggers membrane fusion with a host cell, while “postfusion” describes the significantly rearranged, activated form of gB that occurs after the fusion process has begun,allowing the viral membrane to merge with the host cell membrane. Without wishing to be bound by any theory, it is generally thought that a stabilized, prefusion EBV gB protein will optimize the presentation of highly neutralizing epitope(s) which elicit neutralizing antibodies that are able to block viral entry into epithelial and B cells so to prevent primary EBV infection and the development of infectious mononucleosis (IM).Variants of EBV Glycoprotein B

[0069] The present disclosure provides variants of EBV glycoprotein B (gB) in which at least two mutations (e.g., substitutions) have been introduced in the amino acid sequence relative to the amino acid sequence of the corresponding wild-type EBV gB. The variants of the EBV gB disclosed herein, in some embodiments, are variants of the full-length EBV gB precursor, including a signal peptide at the N terminus, followed by a long ectodomain, and a transmembrane domain and a cytoplasmic tail at the C terminus. Thus, in some embodiments, a variant of an EBV gB disclosed herein comprises an EBV gB ectodomain, an EBV gB transmembrane domain, an EBV gB cytoplasmic tail. The variant of an EBV gB may comprise an EBV gB signal peptide. The mature form of a variant of EBV gB typically does not comprise a signal peptide sequence. However, a nucleotide sequence encoding a signal peptide sequence (e.g., an EBV gB signal peptide sequence) may be present in the artificial nucleic acids described herein encoding the EBV gB polypeptides described herein. In some embodiments, the variants of the EBV gB disclosed herein are variants of the EBV gB ectodomain, optionally preceded by a signal peptide at the N terminus, but without the transmembrane domain and the cytoplasmic tail at the C terminus. Thus, in some embodiments, a variant of an EBV gB disclosed herein comprises an EBV gB ectodomain, but comprises neither an EBV gB transmembrane domain nor an EBV gB cytoplasmic tail.

[0070] The variants of the EBV gB disclosed herein can be from any wild-type EBV gB known in the art or discovered in the future, including, but not limited to, the EBV gB comprising the amino acid sequence of SEQ ID NO: 1. Another exemplary wild-type EBV gB that can be used as the source of the variants of the present disclosure is the EBV subtype identified in southern China and comprising the amino acid sequence under the GenBank accession number QCF57191.1, which is reproduced below (signal peptide: bold and italic, TM: bold; CTD: italic)'.M77? / ? / ?ELAI LZAAZAC / ?£G4QTPEQPAPPATTVQPTATRQQTSFPFRV CELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKI VTNILIYNGWYADSVTNRHEEKFSVESYETDQMDTIYQCYNAVKMTKDGLTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGWLIWTYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNTETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPPAARGSTSAAVLRRRRRNAGNATTPVPPAAPGKSLG TLNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKI NPTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSET MCYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSG NEIHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRAS NVFDLEGIFREYNFQAQNIAGLRKDLDNAVSNGRNQFVDGLGELMDSL GSVGQSITNLVSTVGGLFSSLVSGFISFFKNPFGGMLILVLVVGVVILVI SEYRRTRQMSQQPVQMLYPGIDELAQQHASGEGPGINPISKTELQAIMLALHE QNQEQKRAAQRAAGPSVASRALQAARDRFPGLRRRRYHDPETAAALLGEAET EF (SEQ IDNO: 2).

[0071] In some embodiments, the variants of the EBV gB disclosed herein possess certain beneficial characteristics, such as improved immunogenic properties and / or improved stability in the prefusion conformation, as compared to the corresponding wild-type EBV gB. For example, the variants of the EBV gB disclosed herein may elicit antibodies (e.g., neutralizing antibodies) that are able to block viral entry into epithelial and / or B cells. In some embodiments, the variants of the EBV gB disclosed herein present epitopes for recognition by broadly protecting antibodies and thus, are useful for creating a universal epitope-based vaccine for inducing protection against EBV. Also provided are artificial nucleic acid molecules that encode the variants of the EBV gB disclosed herein.

[0072] The variants of the EBV gB of the present disclosure comprise at least two mutations (e.g., substitutions), typically in the ectodomain, as compared to the amino acid sequence of the corresponding wild-type EBV gB. The introduced amino acid mutations in the variants of the EBV gB of the disclosure include amino acid substitutions, deletions, or additions. In some embodiments, the at least two amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the only mutations introduced in the amino acid sequence of the variants of the EBV gB of the present disclosure areamino acid substitutions relative to the corresponding wild-type EBV gB, and may include conservative and / or non-conservative substitutions.

[0073] Conservative substitutions may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. For example, as summarized in Table 1, the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups shown above.

[0074] In some embodiments, the substitutions may also include non-classical amino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionine P-alanine, GABA and 5-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, P-alanine, fluoro-amino acids, designer amino acids such as P methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general). In some embodiments, amino acid substitutions at a specific amino acid position are chosen based on factors which include, but are not limited to, potential for steric hindrance, charge attraction, charge repulsion, common properties of the amino acid side chain, secondary and / or tertiary structure considerations, and / or frequency of use in respective host cells. A person skilled in the art would understand which factors to consider when designing amino acid substitutions for the variants of the EBV gB disclosed herein.

[0075] The present disclosure is based, in part, on the surprising discovery that introducing at least two cysteine substitutions in certain regions of the EBV gB ectodomain resulted in EBV gBvariants stabilized in the prefusion conformation. Each pair of cysteine substitutions was designed based on analysis of the EBV gB postfusion (experimental structure) and the prefusion (3-D model) structures to identify residues that are in close proximity and located in a region that, when linked by an artificially introduced disulfide bridge, may prevent the structural rearrangement needed to transform the structure from prefusion to postfusion state. Thus, based on a combination of several approaches, including computational design and structure based mutational approach, the inventors have found at least three regions in the EBV gB ectodomain that are important for stabilizing the EBV gB trimer in the prefusion conformation via formation of disulfide bridges. The first region is located at amino acid residues 49-56 of the EBV gB as indexed by reference to the amino acid sequence of SEQ ID NO: 1, the second region is located at amino acid residues 525-529 the EBV gB as indexed by reference to the amino acid sequence of SEQ ID NO: 1, and the third region is located at amino acid residues 631-637 the EBV gB as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Because the amino acid numbering is the same between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2, these three regions correspond to the same regions in the amino acid sequence of SEQ ID NO: 2.

[0076] By identifying these specific regions in the EBV gB ectodomain, the inventors have discovered a strategy for stabilizing EBV gB in a prefusion conformation that requires only minimal alterations to the EBV gB ectodomain. This strategy makes it possible to design EBV gB variants that are not only stabilized in the prefusion conformation but that also share a high level of amino acid sequence identity to the wild-type EBV gB ectodomain. Without intending to be bound by any theory, it is believed that introducing minimal modifications to the EBV gB ectodomain helps to maintain or closely resemble the intact, naturally-occurring EBV gB antigen structure, and to preserve the naturally occurring epitopes of the wild-type EBV gB ectodomain. Accordingly, minimally altered structure is important, and particularly suitable, for vaccines.

[0077] Accordingly, in one aspect, provided herein is a variant of an EBV gB, wherein the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, and wherein the at least two cysteine substitutions are located in two of the three aforementioned regions. In some embodiments, one of the at least two cysteine substitutions is located in the first region located at amino acid residues 49-56 of the EBV gB as indexed by reference to the amino acid sequence of SEQ ID NO: 1 and the other cysteinesubstitution is located in the second region located at amino acid residues 525-529 of the EBV gB as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, one of the at least two cysteine substitutions is located in the first region located at amino acid residues 49-56 of the EBV gB as indexed by reference to the amino acid sequence of SEQ ID NO: 1 and the other cysteine substitution is located in the third region located at amino acid residues 631-637 of the EBV gB as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, one of the at least two cysteine substitutions is located in the second region located at amino acid residues 525-529 of the EBV gB as indexed by reference to the amino acid sequence of SEQ ID NO: 1 and the other cysteine substitution is located in the third region located at amino acid residues 631-637 of the EBV gB as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0078] In some embodiments, the at least two cysteine substitutions in the modified EBV gB ectodomain are the only modifications (e.g., mutations, such as additions, deletions and substitutions) introduced into the EBV gB ectodomain to promote the stabilized prefusion conformation, e.g., relative to the EBV gB ectodomain that does not have the modifications. In some embodiments, the at least two cysteine substitutions in the modified EBV gB ectodomain are the only modifications in the modified EBV gB ectodomain. In some embodiments, the modified EBV gB ectodomain shares at least 93% sequence identity with the unmodified EBV gB ectodomain (e.g., a wild-type EBV gB ectodomain, such as amino acid residues 23-685 of SEQ ID NO: 1 or SEQ ID NO: 2) into which modifications were introduced to generate the modified EBV gB ectodomain. In some embodiments, the modified EBV gB ectodomain shares at least 94% sequence identity with the unmodified EBV gB ectodomain (e.g., a wild-type EBV gB ectodomain, such as amino acid residues 23-685 of SEQ ID NO: 1 or SEQ ID NO: 2) into which modifications were introduced to generate the modified EBV gB ectodomain. In some embodiments, the modified EBV gB ectodomain shares at least 95% sequence identity with the unmodified EBV gB ectodomain (e.g., a wild-type EBV gB ectodomain, such as amino acid residues 23-685 of SEQ ID NO: 1 or SEQ ID NO: 2) into which modifications were introduced to generate the modified EBV gB ectodomain. In some embodiments, the modified EBV gB ectodomain shares at least 96% sequence identity with the unmodified EBV gB ectodomain (e.g., a wild-type EBV gB ectodomain, such as amino acid residues 23-685 of SEQ ID NO: 1 or SEQ ID NO: 2) into which modifications were introduced to generate the modified EBV gBectodomain. In some embodiments, the modified EBV gB ectodomain shares at least 97% sequence identity with the unmodified EBV gB ectodomain (e.g., a wild-type EBV gB ectodomain, such as amino acid residues 23-685 of SEQ ID NO: 1 or SEQ ID NO: 2) into which modifications were introduced to generate the modified EBV gB ectodomain. In some embodiments, the modified EBV gB ectodomain shares at least 98% sequence identity with the unmodified EBV gB ectodomain (e.g., a wild-type EBV gB ectodomain, such as amino acid residues 23-685 of SEQ ID NO: 1 or SEQ ID NO: 2) into which modifications were introduced to generate the modified EBV gB ectodomain. In some embodiments, the modified EBV gB ectodomain shares at least 99% sequence identity with the unmodified EBV gB ectodomain (e.g., a wild-type EBV gB ectodomain, such as amino acid residues 23-685 of SEQ ID NO: 1 or SEQ ID NO: 2) into which modifications were introduced to generate the modified EBV gB ectodomain was generated. In other words, in certain embodiments, the modified gB EBV ectodomain has modifications relative to an unmodified EBV gB ectodomain and shares at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identify with the unmodified EBV gB ectodomain (such as amino acid residues 23-685 of SEQ ID NO: 1 or SEQ ID NO: 2). In some embodiments, the unmodified EBV gB ectodomain is a wild-type EBV gB ectodomain, including, but not limited to the EBV gB ectodomain of SEQ ID NO: 1 or SEQ ID NO: 2. However, any unmodified EBV gB can be modified to generate the modified gB EBV ectodomain and can be aligned and indexed relative to SEQ ID NO: 1 to identify the first, second, and third regions in which the at least two cysteine substitutions are located.

[0079] In some embodiments, the modified gB ectodomain comprising the modifications to promote the stabilized prefusion conformation (e.g., the cysteine substitutions described herein) shares at least 93%, 94%, 95%, 96%, 97%, 98% or 99% (e.g., 98% or 99%) sequence identity with a sequence of an EBV gB ectodomain, e.g., a wild-type EBV gB ectodomain from which the modified EBV gB ectodomain was generated (e.g., residues 23-685 of SEQ ID NO: 1 or SEQ ID NO: 2, and the percentage sequence identity is determined in the absence of any modifications other than those that promote the stabilized prefusion conformation (e.g., the cysteine substitutions described herein). In some embodiments, the modified gB ectodomain comprises modifications (e.g., mutations such as substitutions, deletions or insertions) other than those that promote the stabilized prefusion conformation, but these are not taken into account when determining percentage sequence identity to the sequence of an EBV gB ectodomain, e.g., a wild-type EBV gBectodomain from which the modified EBV gB ectodomain was generated. The stabilized prefusion conformation may be assessed relative to a EBV gB ectodomain that does not have the modifications.

[0080] In some embodiments, the modified EBV gB ectodomain contains a loop between Domain II and Domain III of the ectodomain. In some embodiments, the modified EBV gB ectodomain does not contain a deletion of more than 10 amino acids. In some embodiments, the modified EBV gB ectodomain does not contain a deletion of more than 20 amino acids. In some embodiments, the modified EBV gB ectodomain does not contain a deletion of more than 30 amino acids. In some embodiments, the modified EBV gB ectodomain does not contain a deletion of more than 40 amino acids. In some embodiments, the modified EBV gB ectodomain does not contain a deletion of 50 or more amino acids. For example, the modified EBV gB ectodomain does not contain a deletion of 50 or more amino acids. In some embodiments, the modified EBV gB ectodomain contain amino acid residues 402-452 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified EBV gB ectodomain contain amino acid residues 394-452 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0081] In some embodiments, the cysteine substitution located in the first region of the EBV gB comprises L53C, S54C, or S55C. In some embodiments, the cysteine substitution located in the second region of the EBV gB comprises V525C, S526C, Q527C, or V529C. In some embodiments, the cysteine substitution located in the third region of the EBV gB comprises L632C, I633C, E634C, N635C, or D637C. Accordingly, in some embodiments, the at least two cysteine substitutions comprised in the variant of the EBV gB according to the present disclosure comprise L53C and V525C, L53C and S526C, L53C and Q527C, L53C and V529C, L53C and L632C, L53C and I633C, L53C and E634C, L53C and N635C, L53C and D637C, S54C and V525C, S54C and S526C, S54C and Q527C, S54C and V529C, S54C and L632C, S54C and I633C, S54C and E634C, S54C and N635C, S54C and D637C, S55C and V525C, S55C and S526C, S55C and Q527C, S55C and V529C, S55C and L632C, S55C and I633C, S55C and E634C, S55C and N635C, or S55C and D637C. In some embodiments, the at least two cysteine substitutions comprised in the variant of the EBV gB according to the present disclosure comprise L53C and D637C, S54C and S526C, S55C and N635C, V525C and L632C, S526C and N635C, Q527C and L632C, or Q527C and E634C. In the embodiments where the at least two cysteinesubstitutions comprised in the variant of the EBV gB comprise Q527C and E634C, the variant may further comprise at least two additional cysteine substitutions relative to the EBV gB. It should be noted that all the amino acid numbering referred to in the present disclosure is as indexed by reference to the amino acid sequence of SEQ ID NO: 1 unless indicated otherwise. In some embodiments, all the amino acid numbering referred to in the present disclosure can be as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, all the amino acid numbering referred to in the present disclosure can be as indexed by reference to the amino acid sequence of any wild type EBV gB sequence.

[0082] An EBV gB ectodomain is about 663-amino acid in length. In an exemplary, wild-type EBV gB polypeptide, the gB ectodomain comprises the amino acid sequence as set forth in residues 23-685 of SEQ ID NO: 1. In another exemplary, wild-type EBV gB polypeptide, the gB ectodomain comprises the amino acid sequence as set forth in residues 23-685 of SEQ ID NO: 2. In addition to the at least two cysteine substitutions, the variant of the EBV gB of the present disclosure may further comprise other modifications, such as insertions, deletions, or truncations, in the ectodomain and so may have a length slightly shorter or longer than the ectodomain of the wild-type EBV gB. For example, the variant of the EBV gB disclosed herein may comprise an ectodomain of at least 640 amino acids in length, such as at least 645, at least 650, at least 655, at least 660, or at least 665 amino acids in length, including all values and subranges therebetween. In some embodiments, the modified EBV gB ectodomain of the variant of the EBV gB of the present disclosure is a truncated from of a wild-type EBV gB ectodomain, wherein the wild-type EBV gB ectodomain lacks up to 4, 5, 10, 15, 20, 25, 30, 35 or 40 amino acid residues of a wildtype EBV gB ectodomain.

[0083] Accordingly, in some embodiments, provided herein is a variant of an EBV gB, wherein the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, wherein the at least two cysteine substitutions are located in two regions of the EBV gB selected from a first region located at amino acid residues 49-56 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, a second region located at amino acid residues 525-529 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, and a third region located at amino acid residues 631-637 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified EBV gB ectodomain shares at least 95%, 96%, 97%, 98%, or 99% sequence identity to the ectodomain of the EBV gB. Insome embodiments, if the variant comprises cysteine substitutions Q527C and E634C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, the variant further comprises at least two additional cysteine substitutions relative to the EBV gB. Also provided herein, in some embodiments, is a variant of an EBV gB, wherein the variant comprises a modified EBV gB ectodomain of at least 640 amino acids in length and comprises at least two cysteine substitutions relative to the EBV gB, and wherein the at least two cysteine substitutions are located in two regions of the EBV gB selected from a first region located at amino acid residues 49-56 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, a second region located at amino acid residues 525-529 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, and a third region located at amino acid residues 631-637 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cysteine substitution located in the first region of the EBV gB comprises L53C, S54C, or S55C, the cysteine substitution located in the second region of the EBV gB comprises V525C, S526C, Q527C, or V529C, and the cysteine substitution located in the third region of the EBV gB comprises L632C, I633C, E634C, N635C, or D637C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least two cysteine substitutions comprise L53C and D637C, S54C and S526C, S55C and N635C, V525C andL632C, S526C andN635C, Q527C andL632C, or Q527C andE634C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0084] The variants of the EBV gB disclosed herein may further comprise additional pair(s) (e.g., 1, 2, 3, or 4 pairs) of cysteine substitutions relative to the EBV gB from which the modified EBV gB is derived. For instance, in some embodiments, the variants may comprise one additional pair of cysteine substitutions with one cysteine substitution located in a fourth region of the EBV gB located at amino acid residues 170-177 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 and another cysteine substitution located in a fifth region of the EBV gB located at amino acid residues 560-567 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cysteine substitution located in the fourth region of the EBV gB comprises G172C, L174C, A175C, or G177C. In some embodiments, the cysteine substitution located in the fifth region of the EBV gB comprises N563C or D564C. Accordingly, in some embodiments, the additional pair of cysteine substitutions comprised in the variant of the EBV gB according to the present disclosure comprise G172C and N563C, L174C and N563C, A175C and N563C, G177C and N563C, G172C and D564C, L174C and D564C, A175C and D564C, orG177C and D564C. In some embodiments, the additional pair of cysteine substitutions comprised in the variant of the EBV gB according to the present disclosure comprises G172C and D564C, or L174C and N563C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant of the EBV gB of the present disclosure further comprises at least two, at least four, or at least six further cysteine substitutions relative to the EBV gB from which the modified EBV gB is derived.

[0085] In addition to the five regions of the EBV gB identified by the inventors as being involved in stabilizing the EBV gB trimer in the prefusion conformation as disclosed herein, the inventors also found that cysteine substitutions at certain residues of the EBV gB can also help stabilize the EBV gB trimer in a prefusion conformation. For instance, as described in the examples, cysteine substitutions at amino acid residues 517 and 657 of SEQ ID NO: 1 (i.e., substitutions K517C and L657C), or amino acid residues 581 and 644 of SEQ ID NO: 1 (i.e., substitutions T581C and Y644C), led to increased formation of prefusion EBV gB trimers. Accordingly, in some embodiments, the variants of the EBV gB disclosed herein comprise a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB from which the modified EBV gB is derived, such as a wild-type EBV gB, including, but not limited to, the wild-type EBV gB of SEQ ID NO: 1 or SEQ ID NO: 2, and the at least two cysteine substitutions are selected from L53C, S54C, S55C, R61C, S63C, I90C, G172C, L174C, A175C, G177C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, Q527C, V529C, N563C, D564C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, L632C, I633C, E634C, N635C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, and R675C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Without intending to be bound by any theory, it is understood that the at least two cysteine substitutions form a disulfide bridge in the modified EB V gB ectodomain. In some embodiments, the at least two cysteine substitutions comprise L53C and D637C. In some embodiments, the at least two cysteine substitutions comprise S54C and S526C. In some embodiments, the at least two cysteine substitutions comprise S55C and N635C. In some embodiments, the at least two cysteine substitutions comprise R61C and T621C. In some embodiments, the at least two cysteine substitutions comprise S63C and T621C. In some embodiments, the at least two cysteine substitutions comprise I90C and T630C. In someembodiments, the at least two cysteine substitutions comprise G172C and D564C. In some embodiments, the at least two cysteine substitutions comprise L174C and N563C. In some embodiments, the at least two cysteine substitutions comprise A175C and V529C. In some embodiments, the at least two cysteine substitutions comprise A175C and E634C. In some embodiments, the at least two cysteine substitutions comprise G177C and E634C. In some embodiments, the at least two cysteine substitutions comprise K214C and I633C. In some embodiments, the at least two cysteine substitutions comprise T225C and N635C. In some embodiments, the at least two cysteine substitutions comprise G227C and T591C. In some embodiments, the at least two cysteine substitutions comprise T229C and N606C. In some embodiments, the at least two cysteine substitutions comprise V318C and G477C. In some embodiments, the at least two cysteine substitutions comprise D320C and D478C. In some embodiments, the at least two cysteine substitutions comprise G322C and D478C. In some embodiments, the at least two cysteine substitutions comprise G322C and A480C. In some embodiments, the at least two cysteine substitutions comprise G322C and A482C. In some embodiments, the at least two cysteine substitutions comprise T323C and A482C. In some embodiments, the at least two cysteine substitutions comprise G376C and T624C. In some embodiments, the at least two cysteine substitutions comprise S389C and F463C. In some embodiments, the at least two cysteine substitutions comprise I500C and I660C. In some embodiments, the at least two cysteine substitutions comprise N501C and F661C. In some embodiments, the at least two cysteine substitutions comprise K517C and L657C. In some embodiments, the at least two cysteine substitutions comprise G520C and Y594C. In some embodiments, the at least two cysteine substitutions comprise V525C and L632C. In some embodiments, the at least two cysteine substitutions comprise S526C and N635C. In some embodiments, the at least two cysteine substitutions comprise Q527C and L632C. In some embodiments, the at least two cysteine substitutions comprise Q527C and E634C. In some embodiments, the at least two cysteine substitutions comprise L580C and Y644C. In some embodiments, the at least two cysteine substitutions comprise T581C and Y644C. In some embodiments, the at least two cysteine substitutions comprise T585C and Q669C. In some embodiments, the at least two cysteine substitutions comprise E586C and A668C. In some embodiments, the at least two cysteine substitutions comprise G659C and Y664C. In some embodiments, the atleasttwo cysteine substitutions comprise R675C and A651C. In embodimentswherein the variants of the present disclosure comprise cysteine substitutions G172C and D564C, A175C and V529C, G322C and D478C, or Q527C and E634C, the variants further comprise at least two additional cysteine substitutions relative to the wild-type EBV gB. In some embodiments, the modified EBV gB ectodomain shares at least 95%, 96%, 97%, 98%, or 99% sequence identity to the ectodomain of the EBV gB.

[0086] The variants of the EBV gB disclosed herein may, in some embodiments, comprise more than one pair of cysteine substitutions. For example, the variants of the EBV gB disclosed herein may comprise two pairs, three pairs, four pairs, or more than four pairs of cysteine substitutions.

[0087] In some embodiments, the variants of the EBV gB disclosed herein comprise at least four cysteine substitutions (i.e., at least two pairs of cysteine substitutions) selected from L53C, S54C, S55C, R61C, S63C, I90C, G172C, L174C, A175C, G177C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, Q527C, V529C, N563C, D564C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, L632C, I633C, E634C, N635C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, and R675C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Without intending to be bound by any theory, it is understood that the at least four cysteine substitutions form two disulfide bridges in the modified EBV gB ectodomain.

[0088] In some embodiments, the at least four cysteine substitutions comprise S55C, Q527C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise G172C, Q527C, D564C, and L632C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise S55C, G172C, D564C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0089] In some embodiments, the at least four cysteine substitutions comprise L53C, S55C, N635C, and D637C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise L53C, Q527C, L632C, and D637C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise S55C, G177C, E634C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, theat least four cysteine substitutions comprise S55C, K517C, N635C, and L657C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise S55C, Q527C, E634C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise S55C, L580C, N635C, and Y644C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise S55C, T581C, N635C, and Y644C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise G172C, A175C, V529C, and D564C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise G172C, G322C, A480C, and D564C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise L174C, V318C, G477C, and N563C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise A175C, G322C, A480C, and V529C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise G177C, Q527C, L632C, and E634C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise K517C, Q527C, L632C, and L657C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise Q527C, L580C, L632C, and Y644C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least four cysteine substitutions comprise Q527C, T581C, L632C, and Y644C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0090] In some embodiments, the variants of the EBV gB disclosed herein comprise at least six cysteine substitutions (i.e., at least three pairs of cysteine substitutions) selected from L53C, S54C, S55C, R61C, S63C, I90C, G172C, L174C, A175C, G177C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, Q527C, V529C, N563C, D564C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, L632C, I633C, E634C, N635C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, and R675C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Withoutintending to be bound by any theory, it is understood that the at least six cysteine substitutions form three disulfide bridges in the modified EBV gB ectodomain.

[0091] In some embodiments, the at least six cysteine substitutions comprise S54C, S55C, S526C, Q527C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least six cysteine substitutions comprise S55C, G172C, Q527C, D564C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least six cysteine substitutions comprise S55C, L174C, Q527C, N563C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least six cysteine substitutions comprise S55C, G177C, Q527C, L632C, E634C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least six cysteine substitutions comprise S55C, V318C, G477C, Q527C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least six cysteine substitutions comprise S55C, D320C, D478C, Q527C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least six cysteine substitutions comprise G172C, A175C, G322C, A480C, V529C, and D564C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0092] In some embodiments, the variants of the EBV gB disclosed herein comprise at least eight cysteine substitutions (i.e., at least four pairs of cysteine substitutions) selected from L53C, S54C, S55C, R61C, S63C, I90C, G172C, L174C, A175C, G177C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, Q527C, V529C, N563C, D564C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, L632C, I633C, E634C, N635C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, and R675C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Without intending to be bound by any theory, it is understood that the at least eight cysteine substitutions form four disulfide bridges in the modified EBV gB ectodomain.

[0093] In some embodiments, the at least eight cysteine substitutions comprise S55C, G172C, G322C, D478C, Q527C, D564C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least eight cysteine substitutions comprise S55C, G172C, G322C, A480C, Q527C, D564C, L632C, N635C, as indexed by referenceto the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least eight cysteine substitutions comprise S55C, L174C, V318C, G477C, Q527C, N563C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0094] It has been shown that the hydrophobic residues localized in the putative fusion loops of the EBV gB at amino acid positions 112-113 and 193-196 of SEQ ID NO: 1 can cause the recombinant EBV ectodomains to form aggregates reminiscent of postfusion rosettes observed for class I and II type viral fusion proteins (Backovic et al., Virology, 2007, 368: 102-113). However, substitution of those residues with less hydrophobic amino acids produced trimeric protein and abrogated the ability of the EBV gB ectodomains to form rosettes (Backovic et al., Virology, 2007, 368: 102-113). Accordingly, in some embodiments, the variants of the EBV gB disclosed herein, in addition to the cysteine substitutions described herein elsewhere, further comprise one or more substitutions at amino acid positions W112, Y113, W193, L194, 1195, or W196, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the substitution at amino acid position W112 is W112H. In some embodiments, the substitution at amino acid position Y113 is Y113R. In some embodiments, the substitution at amino acid position W193 is W193R. In some embodiments, the substitution at amino acid position L194 is L194V. In some embodiments, the substitution at amino acid position 1195 is I195E. In some embodiments, the substitution at amino acid position W196 is W196A. In some embodiments, the variants of the EBV gB disclosed herein, in addition to the cysteine substitutions described herein elsewhere, further comprise the substitutions W112H, Y113R, W193R, L194V, I195E, and W196A, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0095] The EBV gB contains a cleavage motif R-X-K / R-R (SEQ ID NO: 3) recognized by the cellular protease furin at R428 to A434 of SEQ ID NO: 1 and it has been shown that deletion of this polybasic furin cleavage site from the EBV gB reduced fusion in both epithelial and B cells as compared to the wild-type EBV gB in a cell-fusion assay (Sorem et al., J. Gen. Virol., 2009, 90(Pt 3):591-595). Accordingly, in some embodiments, the variants of the EBV gB disclosed herein, in addition to the cysteine substitutions described herein elsewhere, further comprise one or more mutations, such as substitutions, insertions, deletions, or truncations, to disrupt the furin cleavage site. In some embodiments, the furin cleavage site is disrupted by inserting one or more amino acids into the region of the EBV gB corresponding to amino acid residues 428-434 of SEQ ID NO: 1 so that the cleavage motif R-X-K / R-R (SEQ ID NO: 3) is no longer present. In someembodiments, the furin cleavage site is disrupted by deleting one or more amino acids from the region of the EBV gB corresponding to amino acid residues 428-434 of SEQ ID NO: 1 so that the cleavage motif R-X-K / R-R (SEQ ID NO: 3) is no longer present. In some embodiments, the furin cleavage site is disrupted by substituting one or more amino acids in the region of the EBV gB corresponding to amino acid residues 428-434 of SEQ ID NO: 1 so that the cleavage motif R-X-K / R-R (SEQ ID NO: 3) is no longer present. In some embodiments, the furin cleavage site of the EBV gB is replaced by a linker, such as a GS linker. In some embodiments, amino acid residues 428-434 of the variants of the present disclosure, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, comprise the amino acid sequence GGGGSGS (SEQ ID NO: 4). In some embodiments, amino acid residues 428-434 of the variants of the present disclosure, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, comprise the amino acid sequence GGSGSGS (SEQ ID NO: 5).

[0096] Wild-type EBV gB comprises a signal peptide at the N-terminus (e.g., corresponding to amino acids 1-22 of SEQ ID NO: 1), which directs transport of the gB during production and generally is not present in the final polypeptide that is, for instance, used in a vaccine. Accordingly, in some embodiments, the variants of the EBV gB of the present disclosure do not comprise a signal peptide. In other embodiments, the variants of the EBV gB of the present disclosure comprise a signal peptide that is native to the wild-type EBV gB and comprises the amino acids sequence as set forth in residues 1-22 of SEQ ID NO: 1, or the amino acids sequence MTRRRVLSVVVLLAALACRLGA (SEQ ID NO: 6). In some embodiments, the variants of the EBV gB of the present disclosure comprise a signal peptide that is heterologous to the wild-type EBV gB, such as the Ig Kappa signal peptide having the amino acids sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 7). Any signal peptide known in the art that can direct transport of the gB during production can be used. For recombinant production of the variants of the EBV gB of the disclosure in insect cells, for instance, any signal peptides from both mammals and viruses can be used to guide protein secretion in insect cells.

[0097] In some embodiments, the variants of the EBV gB disclosed herein, comprise a multimerization domain, such as a trimerization domain. In some embodiments, the multimerization domain, such as a trimerization domain is located on the C-terminus of the variant EBV gB. A trimerization domain is a peptide segment that allows for the polypeptide to form a trimer (e.g., upon expression). In some embodiments, the trimerization domain comprises or is T4bacteriophage fibritin foldon (Fd) trimerization domain, which is at the C-terminal domain of T4 fibritin. In some embodiments, the trimerization domain comprises the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 8). Other trimerization domains known in the art can also be used.

[0098] In some embodiments, the variants of the EBV gB disclosed herein comprise a StrepII-tag, a tag sequence, and / or a protease cleavage site on the C-terminus to facilitate recombinant production and purification of the variants. In some embodiments, the variants disclosed herein comprise a StrepII-tag comprising the amino acid sequence WSHPQFEK (SEQ ID NO: 9) on the C-terminus. In some embodiments, the variants disclosed herein comprise a tag sequence, such as a His tag comprising the amino acid sequence HHHHHH (SEQ ID NO: 10) or HHHHHHH (SEQ ID NO: 11), a FLAG tag comprising the amino acid sequence DYKDDDDK (SEQ ID NO: 12), or a C-tag comprising the amino acid sequence EPEA (SEQ ID NO: 13) on the C-terminus. In some embodiments, the variants disclosed herein comprise a protease cleavage site, such as a human rhinovirus (HRV) 3C protease cleavage site comprising the amino acid sequence LEVLFQ / GP (SEQ ID NO: 14), a Factor X protease cleavage site comprising the amino acid sequence IE / DGR (SEQ ID NO: 15), or a thrombin cleavage site comprising the amino acid sequence LVPRGS (SEQ ID NO: 16), on the C-terminus.

[0099] In some embodiments, the variants of the EBV gB disclosed herein comprise one or more linkers, such as glycine-serine (GS) flexible linkers which have been shown to improve folding and stability in several fusion protein examples. For instance, in some embodiments, the variants of the EBV gB disclosed herein may comprise a GGS linker before the trimerization domain (e.g., foldon), a GRS linker between the trimerization domain (e.g., foldon) and the HRV 3C protease cleavage site, a G linker between the HRV 3C protease cleavage site and the StrepII-tag, and / or a GS linker between the StrepII-tag and the His-tag. Other linkers known in the art, such as those described in Chen et al., Adv. Drug Deliv. Rev., 2012, 65(10): 1357-1369 (incorporated herein by reference), can also be used.

[0100] In some embodiments, the variants of the EBV gB disclosed herein further comprise one or more substitutions that results in one or more N-linked glycosylation sites being introduced. Without wishing to be bound by any theory, adding additional N-linked glycosylation site(s) can add additional glycan(s) to mask non-neutralizing epitopes presented in the region, and promote a neutralizing response by the variants. In some embodiments, the N-linked glycosylation sitecomprises a consensus sequence of NxS / Ty (SEQ ID NO: 17), in which N is Asparagine, S / T is a Serine or Threonine residue, and x and y are any residue except Proline (P).

[0101] In some embodiments, relative to a corresponding wild-type EBV gB, the at least one N-linked glycosylation site introduced in the variants of the EBV gB of the disclosure is generated by introducing one or more substitutions at amino acid positions 32, 43, 130, 265, 361, 450, 533, 545, or 611, and / or amino acid positions 67 and 69, 112 and 114, 150 and 152, 153 and 155, 159 and 161, 167 and 169, 176 and 178, 184 and 186, 264 and 266, 299 and 301, 336 and 338, 389 and 391, 403 and 405, 442 and 443, 532 and 534, 548 and 550, 593 and 595, 634 and 636, 639 and 641, 647 and 649, or 677 and 679, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitution A32N. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitution Q43N. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitution Y130N. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitution R265N. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitution R361N. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitution L450N. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitution Q533N. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitution P545N. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitution F611N. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions Q67N and P69S. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions Hl 12N and Al 14S. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions D150N and L152T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions T153N and V155T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions R159N and G161T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions N167 and K169S. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions N176 and V178T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions Q184N and E186T. In some embodiments, the at least one N-linkedglycosylation site is generated by amino acid substitutions N264 and G266T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions H299N and Q301T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions L336N and D338S. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions S389N and A391T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions T403N and P405T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions P442N and P443 A. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions N532 and A534S. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions E548N and M550T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions Q593N and Y595T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions E634N and I636T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions A639N and L641T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions D647N and Q649T. In some embodiments, the at least one N-linked glycosylation site is generated by amino acid substitutions D677N and E679T.Exemplary Variants ofEBVgB

[0102] In some embodiments, relative to a corresponding wild-type EBV gB (e.g., SEQ ID NO: 1 or SEQ ID NO: 2), the variants of the EBV gB provided herein comprise any of the following combinations of amino acid substitutions listed in the following table, as indexed by reference to the amino acid sequence of SEQ ID NO: 1 :

[0103] As shown herein, such amino acid substitutions can stabilize the variants of the EBV gB in a prefusion (closed) conformation, as measured by, for instance, negative stain electron microscopy, as compared to the corresponding wild-type EBV gB without the amino acid substitutions.

[0104] In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions S55C and 635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions G172C and D564C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions Q527C and L632C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions S55C, Q527C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions S55C, G172C, D564C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gBprovided herein comprise amino acid substitutions G172C, D564C, Q527C, and L632C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions S55C, G172C, Q527C, D564C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0105] In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions L53C and D637C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions K517C and L657C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions Q527C and E634C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions T581 and Y644C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB provided herein comprise amino acid substitutions S54C, S55C, S526C, Q527C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0106] In some embodiments, the variants of the EBV gB disclosed herein are derived from the wild-type EBV gB of SEQ ID NO: 1, but with substitutions at amino acid residues 112-113 and 193-196 (fusion loop) and 428-434 (furin cleavage site) described herein elsewhere, as well as modifications at the C-terminus, including truncation from R686 to F868 to remove the transmembrane domain, the cytoplasmic tail, and the membrane proximal domains and addition of a T4-Foldon trimerization domain (G688 to L714 of SEQ ID NO: 18), a StrepII-tag (W729 to K736 of SEQ ID NO: 18), a His-tag (H739 to H746 of SEQ IDD NO: 18), linkers, and a HRV 3C protease cleavage site (L718 to P725 of SEQ ID NO: 18). The amino acid sequence of this base sequence (construct rEBVgB_G685) is reproduced below:MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRV CELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKI VTNILIYNGHRADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQHWQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPSAARGSTPAAVLGGGGSGSGNATTPVPPTAPGKSLGT LNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKIN PTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETM CYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNE IHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRASNV FDLEGIFREYNFQAQNIAGLRKDLDNAVSNGGSGYIPEAPRDGQAYVRKD GEWVLLSTFLGRSLEVLFQGPGSAWSHPQFEKGSHHHHHHHH (SEQ ID NO: 18),

[0107] In some embodiments, the variants of the EB V gB, such as the variants of the EBV gB ectodomain, are derived from the wild-type EBV gB of SEQ ID NO: 2, but with substitutions at amino acid residues 112-113 and 193-196 (fusion loop) and 428-434 (furin cleavage site) described herein elsewhere and substitutions S405P, P410T, P416S, A444T, and D679E to remove potential human cross reactivity (HPX), as well as modifications at the C-terminus, including truncation from R686 to F868 to remove the transmembrane domain, the cytoplasmic tail, and the membrane proximal domains and addition of a T4-Foldon trimerization domain (G688 to L714 of SEQ ID NO: 19), a StrepII-tag (W729 to K736 of SEQ ID NO: 19), a His-tag (H739 to H746 of SEQ IDD NO: 19), a C-tag (E750-A753 of SEQ ID NO: 19), linkers, and a HRV 3C protease cleavage site (L718 to P725 of SEQ ID NO: 19). The amino acid sequence of this base sequence (construct rEBV-gB_NPCT074) is reproduced below:MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRV CELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKI VTNILIYNGHRADSVTNRHEEKFSVESYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNTETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSPPPSSTSPPAPSAARGSTSAAVLGGSGSGSGNATTPVPPTAPGKSLGT LNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKINPTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETM CYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNE IHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRASNV FDLEGIFREYNFQAQNIAGLRKDLENAVSNGGSGYIPEAPRDGQAYVRKD GEWVLLSTFLGRSLEVLFQGPGSAWSHPQFEKGSHHHHHHHHGAAEPEA(SEQ ID NO: 19).

[0108] Representative variants of the EBV gB generated based on the base sequence of SEQ ID NO: 18 according to the present disclosure may have the amino acid sequences set forth below with the cysteine substitutions in bold and double underlined.Construct rEBVgB_G685_S55C-N635C (Construct rEBVgB_G685 with amino acid substitutions S55C andN635C):MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRV CELSCHGDLFRF S SDIQCPSFGTRENHTEGLLMVFKDNIIP YSFKVRS YTKI VTNILIYNGHRADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPSAARGSTPAAVLGGGGSGSGNATTPVPPTAPGKSLGT LNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKIN PTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETM CYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNE IHVYNDYHHFKTIELDGIATLQTFISLNTSLIECIDFASLELYSRDEQRASNV FDLEGIFREYNFQAQNIAGLRKDLDNAVSNGGSGYIPEAPRDGQAYVRKD GEWVLLSTFLGRSLEVLFQGPGSAWSHPQFEKGSHHHHHHHH (SEQ ID NO: 20)Construct rEBVgB_G685_Q527C-L632C (Construct rEBVgB_G685 with amino acid substitutions Q527C and L632C):MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRV CELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKIVTNILIYNGHRADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPSAARGSTPAAVLGGGGSGSGNATTPVPPTAPGKSLGT LNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKIN PTTVMSSIYGI<AVAAI<RLGDVISVSCCVPVNQATVTLRI<SMRVPGSETM CYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNE IHVYNDYHHFKTIELDGIATLQTFISLNTSCIENIDFASLELYSRDEQRASN VFDLEGIFREYNFQAQNIAGLRKDLDNAVSNGGSGYIPEAPRDGQAYVRK DGEWVLLSTFLGRSLEVLFQGPGSAWSHPQFEKGSHHHHHHHH (SEQ ID NO: 21)Construct rEBVgB_G685_Q527C-E634C (Construct rEBVgB_G685 with amino acid substitutions Q527C and E634C):MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRV CELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKI VTNILIYNGHRADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPSAARGSTPAAVLGGGGSGSGNATTPVPPTAPGKSLGT LNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKIN PTTVMSSIYGKAVAAKRLGDVISVSCCVPVNQATVTLRKSMRVPGSETM CYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNE IHVYNDYHHFKTIELDGIATLQTFISLNTSLICNIDFASLELYSRDEQRASN VFDLEGIFREYNFQAQNIAGLRKDLDNAVSNGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSAWSHPQFEKGSHHHHHHHH (SEQ ID NO: 22)Construct rEBVgB_G685_K517C-L657C (Construct rEBVgB_G685 with amino acid substitutions K517C and L657C):MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRV CELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKI VTNILIYNGHRADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPSAARGSTPAAVLGGGGSGSGNATTPVPPTAPGKSLGT LNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKIN PTTVMSSIYGKAVAACRLGDVISVSQCVPVNQATVTLRKSMRVPGSETM CYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNE IHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRASNV FDCEGIFREYNFQAQNIAGLRKDLDNAVSNGGSGYIPEAPRDGQAYVRK DGEWVLLSTFLGRSLEVLFQGPGSAWSHPQFEKGSHHHHHHHH (SEQ ID NO: 23)Construct rEBVgB_G685_L53C-D637C (Construct rEBVgB_G685 with amino acid substitutions L53C and D637C):MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRV CECS SHGDLFRF S SDIQCP SFGTRENHTEGLLMVFKDNIIP YSFKVRS YTKI VTNILIYNGHRADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPSAARGSTPAAVLGGGGSGSGNATTPVPPTAPGKSLGTLNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKIN PTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETM CYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNE IHVYNDYHHFKTIELDGIATLQTFISLNTSLIENICFASLELYSRDEQRASNV FDLEGIFREYNFQAQNIAGLRKDLDNAVSNGGSGYIPEAPRDGQAYVRKD GEWVLLSTFLGRSLEVLFQGPGSAWSHPQFEKGSHHHHHHHH (SEQ ID NO: 24)Construct rEBVgB_G685_T581C-Y644C (Construct rEBVgB_G685 with amino acid substitutions T581C and Y644C):MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRV CELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKI VTNILIYNGHRADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPSAARGSTPAAVLGGGGSGSGNATTPVPPTAPGKSLGT LNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKIN PTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETM CYSRPLVSFSFINDTKTYEGQLGTDNEIFLCKKMTEVCQATSQYYFQSGN EIHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELCSRDEQRASN VFDLEGIFREYNFQAQNIAGLRKDLDNAVSNGGSGYIPEAPRDGQAYVRK DGEWVLLSTFLGRSLEVLFQGPGSAWSHPQFEKGSHHHHHHHH (SEQ ID NO: 25)Construct rEBVgB_G685_S54C-S526C_S55C-N635C_Q527C-L632C (Construct rEBVgB_G685 with amino acid substitutions S54C, S526C, S55C, N635C, Q527C, and L632C):MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRV CELCCHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKI VTNILIYNGHRADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRTRTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPSAARGSTPAAVLGGGGSGSGNATTPVPPTAPGKSLGT LNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKIN PTTVMSSIYGKAVAAKRLGDVISVCCCVPVNQATVTLRKSMRVPGSETM CYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNE IHVYNDYHHFKTIELDGIATLQTFISLNTSCIECIDFASLELYSRDEQRASN VFDLEGIFREYNFQAQNIAGLRKDLDNAVSNGGSGYIPEAPRDGQAYVRK DGEWVLLSTFLGRSLEVLFQGPGSAWSHPQFEKGSHHHHHHHH (SEQ ID NO: 26)Construct rEBVgB_G685_G172C-D564C_S55C-N635C_Q527C-L632C (Construct rEBVgB_G685 with amino acid substitutions G172C, D564C, S55C, N635C, Q527C, and L632C):MTRRRVLSVVVLLAALACRLGAQTPEQPAPPATTVQPTATRQQTSFPFRV CELSCHGDLFRF S SDIQCPSFGTRENHTEGLLMVFKDNIIP YSFKVRS YTKI VTNILIYNGHRADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDG LTRVYVDRDGVNITVNLKPTCGLANGVRRYASQTELYDAPGRVEATYRT RTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADS FHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQH WQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKT MHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELT TPTSSPPSSPSPPAPSAARGSTPAAVLGGGGSGSGNATTPVPPTAPGKSLGT LNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKIN PTTVMSSIYGI<AVAAI<RLGDVISVSCCVPVNQATVTLRI<SMRVPGSETM CYSRPLVSFSFINCTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNE IHVYNDYHHFKTIELDGIATLQTFISLNTSCIECIDFASLELYSRDEQRASN VFDLEGIFREYNFQAQNIAGLRKDLDNAVSNGGSGYIPEAPRDGQAYVRK DGEWVLLSTFLGRSLEVLFQGPGSAWSHPQFEKGSHHHHHHHH (SEQ ID NO: 27)

[0109] Accordingly, in some embodiments, the variants of the EBV gB of the present disclosure comprise an amino acid sequence having at least about 90%, such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, including all values and subranges therebetween, sequence identity to the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. In some embodiments, the variants of the EBV gB of the present disclosure comprise or consist of the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

[0110] Similar to wild-type EBV gB, the variants of the EBV gB disclosed herein are capable of forming a trimeric gB complex. Thus, in some embodiments, provided herein is a trimeric EBV gB complex comprising three copies of the variant of the EBV gB according to the present disclosure (i.e., three copies of a single variant EBV gB as described herein). In certain embodiments, the trimeric EBV gB complex of the disclosure has improved stability in the prefusion conformation as compared to a trimeric EBV gB complex of a corresponding wild-type EBV gB without the modifications. Conformation stability can be measured using any methods known in the art. In some embodiments, stability in the prefusion conformation is measured by negative stain electron microscopy.[OHl] In some embodiments, the trimeric EBV gB complex of the disclosure is more immunogenic (e.g., elicits a greater antibody (such as neutralizing antibody) response) as compared to a trimeric EBV gB complex of a corresponding wild-type EBV gB without the modifications. In some embodiments, the trimeric EBV gB complex of the present disclosure has a comparable immunogenicity (e.g. elicits a comparable antibody (such as neutralizing antibody) response) as a trimeric EBV gB complex of a corresponding wild-type EBV gB without the modifications. Immunogenicity can be measured using any methods known in the art.

[0112] While the variants of the EBV gB with particular amino acid substitutions at exemplary amino acid positions are provided herein, the present disclosure also contemplates amino acid substitutions in close proximity to the specified amino acid positions. Therefore, in some embodiments, the present disclosure also comprises amino acid positions within three residues (e.g., within one or two residues) of an amino acid position specified herein. For example, the disclosure of a substitution at amino acid position 55 of the EBV gB, as indexed by reference tothe amino acid sequence of SEQ ID NO: 1, may also include a substitution at position 52, 53, 54, 56, 57, or 58, respectively.

[0113] This is illustrated by the exemplified variants of the EBV gB described herein. For instance, cystine substitutions at amino acid positions 53 and 637, or amino acid positions 55 and 635, both resulted in disulfide bridge formation. Likewise, cystine substitutions at amino acid positions 61 and 621, or amino acid positions 63 and 621, both resulted in disulfide bridge formation. Similarly, cystine substitutions at amino acid positions 172 and 564, or amino acid positions 174 and 563, both resulted in disulfide bridge formation.Nucleic Acid Construction and Expression

[0114] The present disclosure further provides artificial nucleic acid molecules encoding the variants of the EBV gB disclosed herein. The nucleic acids may be DNA or RNA and may be wholly or partially synthetic or recombinant. The variants of the EBV gB provided herein can be encoded by DNA sequences (e.g., synthesized by standard methods known in the art). These DNA sequences may be subsequently cloned and expressed in a recombinant host system using a suitable vector. The variants of the EBV gB provided herein can also be encoded by a RNA, such as messenger RNA (mRNA), sequences. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence and encompasses an RNA molecule (e.g., mRNA) with the specified sequence in which U, or a derivative thereof, such as pseudouridine, is substituted for T, unless context requires otherwise. Other nucleotide derivatives or modified nucleotides can be incorporated into the artificial nucleic acid molecules encoding the disclosed variants of the EBV gB. The synthesized DNA or mRNA sequences encoding the variants of the EBV gB of the disclosure can be codon-optimized so that expression of the encoded protein is improved and optimized for a particular expression system. Any codon optimization algorithms known in the art can be used to generate codon-optimized nucleic acid sequences.

[0115] To express the variants of the EBV gB of the present disclosure, suitable recombinant host cells include, but are not limited to, 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 BTLTN-5B1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include, but are not limited to, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells, typically transformed by sheared adenovirus type 5DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, but are not limited to, for example, chicken embryonic stem cells (e.g., EBx® cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts, and duck cells. Suitable insect cell expression systems, such as baculovirus-vectored systems, are known to those of skill in the art and described in, for instance, Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, for example, Invitrogen (San Diego, CA). Avian cell expression systems are also known to those of skill in the art and described in, for example, U.S. Pat. Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668. Similarly, bacterial and mammalian cell expression systems are also known in the art and described in, for example, Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

[0116] In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6™ cell). In some embodiments, the host cell is a SF9 cell of Spodoptera frugiperda. See, U.S. Patent No. 6,103,526, which is hereby incorporated by reference in its entirety. In some embodiments, the host cell is a SF9 cell of Spodoptera frugiperda which has been infected with a baculovirus vector (e.g., Autographa californica nuclear polyhedrosis virus). In some embodiments, the host cell is a CHO cell.

[0117] 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 nonmammalian species). For example, for expression in insect cells, a suitable baculovirus expression vector, such as pFastBac (Invitrogen), 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., CHO cells) can be used.

[0118] The variants of the EBV gB of the disclosure can be purified using any suitable methods. For example, methods for purifying recombinant polypeptides 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 well-known in the art. Suitable purification schemes can be created using two or more of these or other suitable methods. If desired, the variants of the present disclosure can include a “tag” that facilitates purification, such as an epitope tag or a histidine (His) tag, as exemplified in the examples. Such tagged polypeptides can conveniently be purified, for example from conditioned media, by chelating chromatography or affinity chromatography.

[0119] Purified polypeptides can be analyzed by spectroscopic methods known in the art, such as circular dichroism spectroscopy, Fourier-transform infrared spectroscopy, NMR spectroscopy, or X-ray crystallography, to investigate the presence of desired structures like helices and beta sheets. ELISA, Octet and FACS and the like can be used to investigate binding of the variants of the EBV gB of the disclosure to the antibodies. Negative stain electron microscopy can be used to investigate the conformation of trimers (e.g., prefusion or postfusion) formed with the variants of the EBV gB disclosed herein. Thus, variants of the EBV gB according to the disclosure having a desired conformation (e.g., stabilized prefusion conformation) can be selected.

[0120] Accordingly, in some embodiments, provided herein is an artificial nucleic acid encoding a variant of the EBV gB described herein. The artificial nucleic acids of the disclosure can be a DNA or a RNA, such as a messenger RNA (mRNA). In some embodiments, the artificial nucleic acids of the disclosure are DNA molecules. In some embodiments, the artificial nucleic acids of the disclosure are RNA molecules. In certain embodiments, the artificial nucleic acids of the disclosure are mRNA molecules.

[0121] Also provided herein are vectors comprising an artificial nucleic acid molecule (e.g., mRNAs) disclosed herein. The RNA sequences encoding a protein of interest (e.g., mRNA encoding a variant of the EBV gB) can be cloned into a number of types of vectors. For example, the nucleic acids can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest can include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0122] In certain embodiments, the vector can be used to express mRNA in a host cell. In various embodiments, the vector can be used as a template for in vitro transcription (IVT). The construction of optimally translated IVT mRNA suitable for therapeutic use is disclosed in detailin Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0123] In some embodiments, the vectors disclosed herein can comprise at least the following, from 5' to 3': an RNA polymerase promoter; a polynucleotide sequence encoding a 5' UTR; a polynucleotide sequence encoding an ORF; a polynucleotide sequence encoding a 3' UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein may comprise a polynucleotide sequence encoding a poly(A) sequence and / or a polyadenylation signal.

[0124] A variety of RNA polymerase promoters are known. In some embodiments, the promoter can be a SP6 RNA polymerase promoter. Other useful promoters can include, but are not limited to, T3 and T7 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known.

[0125] Also disclosed herein are host cells (e.g., mammalian cells, e.g., human cells) comprising an artificial nucleic acid (e.g., DNA or RNA) or a vector disclosed herein.

[0126] Polynucleotides can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg, Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. (2001). Hum Gene Ther.12(8):861-70, or the TransIT-RNA transfection Kit (Minis, Madison, WI).

[0127] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0128] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the mRNA sequence in the host cell, a variety of assays may be performed.Epstein-Barr Virus-Like Particle (VLP)

[0129] In some embodiments, the variants of the EBV gB disclosed herein are incorporated into viral-like particle (VLP) vectors. VLPs generally comprise a viral polypeptide(s) typically derived from a structural protein(s) of a virus, such as the EBV gB. Preferably, the VLPs are not capable of replicating. In some embodiments, the VLPs can lack the complete genome of a virus or comprise a portion of the genome of a virus. In some embodiments, the VLPs are not capable of infecting a cell. In some embodiments, the VLPs express on their surface one or more of viral (e.g., virus surface glycoprotein) or non-viral (e.g., antibody or protein) targeting moieties known to one skilled in the art.

[0130] In some embodiments, the EBV VLPs of the present disclosure comprise, in addition to the variant of the EBV gB, one or more additional EBV glycoproteins. In some embodiments, the one or more additional EBV glycoproteins comprise the glycoprotein complex gH / gL. In some embodiments, the one or more additional EBV glycoproteins comprise gp42. In some embodiments, the one or more additional EBV glycoproteins comprise gp350. In some embodiments, the one or more additional EBV glycoproteins comprise gp220. In some embodiments, the one or more additional EBV glycoproteins comprise a combination of any of gHgL, gp42, gp350, and / or gp220. In some embodiments, the EBV VLPs of the present disclosure comprise, in addition to the variant of the EBV gB, one or more T cell antigens selected from BZLF1, BMRF1, EBNA3a, EBNA3b, EBNA3c, LMP2, BRFL1, BMLF1, and / or EBNA-1. In some embodiments, the one or more T cell antigens comprise BZLF1. In some embodiments, the one or more T cell antigens comprise BMRF1. In some embodiments, the one or more T cell antigens comprise EBNA3a. In some embodiments, the one or more T cell antigens comprise EBNA3b. In some embodiments, the one or more T cell antigens comprise EBNA3c. In some embodiments, the one or more T cell antigens comprise LMP2. In some embodiments, the one or more T cell antigens comprise BRFL1. In some embodiments, the one or more T cell antigens comprise BMLF1. In some embodiments, the one or more T cell antigens comprise EBNA-1. In some embodiments, the one or more T cell antigens comprise a combination of BZLF1, BMRF1, EBNA3a, EBNA3b, EBNA3c, LMP2, BRFL1, BMLF1, and / or EBNA-1.

[0131] Production of EBV VLPs is known in the art and will be readily apparent to persons of skill upon reading the present disclosure. For example, EBV VLPs may be produced by transfection of host cells with plasmids encoding the variant of the EBV gB of the presentdisclosure, and other EBV glycoproteins, if present. To give but one example, a suitable host cell includes a human cell (e.g., HEK293T). After incubation of the transfected cells for an appropriate time to allow for protein expression (such as for approximately 72 hours), VLPs may be isolated from cell culture supernatants. In some embodiments, the EBV VLPs of the present disclosure may be used in immunogenic compositions described herein or as EBV vaccines to elicit a broadly neutralizing immune response against the EBV infection.Artificial Messenger Ribonucleic Acids

[0132] Also provided herein, in some embodiments, are artificial mRNAs encoding a variant of the EBV gB comprising any of the cysteine substitutions as disclosed herein elsewhere. Such artificial mRNAs can be useful for, for instance, producing mRNA vaccines. To ensure proper processing of the encoded protein in vivo, the variants of the EBV gB encoded by the artificial mRNAs of the present disclosure are variants of the full-length EBV gB precursor and thus, comprise the N-terminal signal peptide and the C-terminal transmembrane domain and cytoplasmic tail but with any of the cysteine substitutions disclosed herein elsewhere in the ectodomain.

[0133] To increase stabilization, in some embodiments, the variants of the EBV gB encoded by any of the artificial mRNAs disclosed herein further comprise at least two cysteine substitutions comprise Y198C and S712C and / or I771C and H802C, as index by reference to the amino acid sequence of SEQ ID NO: 1. Without wishing to be bound by any theory, the cysteine substitutions Y198C and S712C are thought to link one of the fusion loops with the membrane proximal helix and the cysteine substitutions 1771C and H802C are thought to make an inter protomer disulfide bridge between two helices in the cytoplasmic domain. Accordingly, in some embodiments, the variants of the EBV gB encoded by any of the artificial mRNAs disclosed herein further comprise at least two cysteine substitutions comprise Y198C and S712C, as index by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB encoded by any of the artificial mRNAs disclosed herein further comprise at least two cysteine substitutions comprise I771C and H802C, as index by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants of the EBV gB encoded by any of the artificial mRNAs disclosed herein further comprise cysteine substitutions Y198C, S712C, I771C, and H802C, as index by reference to the amino acid sequence of SEQ ID NO: 1.

[0134] It has been shown that mutating the endoplasmic reticulum retention signal RRRR (SEQ ID NO: 28) located at amino acid residues 836-839 of SEQ ID NO: 1 to RTTR (SEQ ID NO: 29) enhanced presentation of the EB V gB at the cell membrane, which could lead to enhanced gB immunogenicity (Haan et al., Virology, 2001, 290:10-114). Accordingly, in some embodiments, in addition to the cysteine substitutions disclosed herein, the variants of the EBV gB encoded by any of the artificial mRNAs disclosed herein further comprises substitutions R837T and R838T, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0135] Representative mRNA sequences encoding variants of the EBV gB according to the present disclosure may have the amino acid sequences set forth below.Construct EBVgB_RTTR_L174C-N563C_S55C-N635C_Q527C-L632C_Y198C- S712C:atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggctgtgccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctgtaggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggc gacctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccccaccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgt tgcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctac tcccggcccctggtgagcttctcttttatttgtgacaccaagacctacgagggccagctgggcaccgacaacgagatc ttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgt gtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgttt gatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgtgtaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaaccc cttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagac agatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcgag ggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagga gcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggaca ggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgagac agagttctgataa (SEQ ID NO: 33)Construct EBVgB mRNA RTTR L 174C-N563C S55C-N635C Q527C-L632C : atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggctgtgccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctgctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggc gacctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccc caccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgt tgcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctac tcccggcccctggtgagcttctcttttatttgtgacaccaagacctacgagggccagctgggcaccgacaacgagatc ttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgt gtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgttt gatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacc ccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccaga cagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcga gggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagg agcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggac aggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgaga cagagttctgataa (SEQ ID NO: 34)Construct EBVgB_mRNA_RRRR_L174C-N563C_S55C-N635C_Q527C- L632C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggctgtgccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggccagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggc gacctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccc caccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgt tgcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctac tcccggcccctggtgagcttctcttttatttgtgacaccaagacctacgagggccagctgggcaccgacaacgagatc ttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgt gtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgttt gatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacc ccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccaga cagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcga gggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagg agcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggac aggttccccggcctgagacggaggagataccacgaccccgaaaccgccgccgccctgctgggcgaggccgaga cagagttctgataa (SEQ ID NO: 35)Construct EBVgB_mRNA_RTTR_D320C-D478C_S55C-N635C_Q527C- L632C_Y198C-S712C:atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagagaaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggcctggccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctgtaggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgacctgtgaggggacctctagctttgtgacca acaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgaga agtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctgc tgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacca gcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctgggc ggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcacc ctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggctg tctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccccac caccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgttgc gtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctactcc cggcccctggtgagcttctcttttatcaacgacaccaagacctacgagggccagctgggcaccgacaacgagatctt cctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgtg tacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaatac cagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgtttg atctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataacgc cgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccagtc tatcacaaacctggtgtgtaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacccct tcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagaca gatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcgagg gccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccaggag cagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggacaggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgagaca gagttctgataa (SEQ ID NO: 36)Construct EBVgB_mRNA_RTTR_D320C-D478C_S55C-N635C_Q527C-L632C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggcctggccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgacctgtgaggggacctctagctttgtgacca acaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgaga agtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctgc tgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacca gcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctgggc ggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcacc ctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggctg tctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccccac caccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgttgc gtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctactcc cggcccctggtgagcttctcttttatcaacgacaccaagacctacgagggccagctgggcaccgacaacgagatctt cctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgtg tacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaatac cagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgtttg atctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataacgc cgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccagtctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaaccc cttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagac agatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcgag ggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagga gcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggaca ggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgagac agagttctgataa (SEQ ID NO: 37)Construct EBVgB_mRNA_RRRR_D320C-D478C_S55C-N635C_Q527C-L632C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggcctggccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgacctgtgaggggacctctagctttgtgacca acaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgaga agtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctgc tgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacca gcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctgggc ggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcacc ctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggctg tctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccccac caccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgttgc gtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctactcc cggcccctggtgagcttctcttttatcaacgacaccaagacctacgagggccagctgggcaccgacaacgagatcttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgtg tacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaatac cagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgtttg atctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataacgc cgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccagtc tatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaaccc cttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagac agatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcgag ggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagga gcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggaca ggttccccggcctgagacggaggagataccacgaccccgaaaccgccgccgccctgctgggcgaggccgagac agagttctgataa (SEQ ID NO: 38)Construct EBVgB_mRNA_RTTR_V318C-G477C L174C-N563C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagcagccacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagaga gaaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacacca agatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctcc gtggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggc ctgaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggctgtgccaac ggcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagg accaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccgg ccagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttcca cgtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcct ggacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagac cttcgacagcaccatcgcaactgagactggcaagtccatccacttttgtaccgacgaggggacctctagctttgtgac caacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacga gaagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcct gctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaac cagcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctgg gcggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcaccctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgtgt gacctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccc caccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtccca gtgcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgcta ctcccggcccctggtgagcttctcttttatttgtgacaccaagacctacgagggccagctgggcaccgacaacgagat cttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacg tgtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagcctgatcgagaacatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgtt tgatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacc ccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccaga cagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcga gggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagg agcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggac aggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgaga cagagttctgataa (SEQ ID NO: 39)Construct EBVgB_mRNA_RTTR_V318C-G477C_L174C-N563C_S55C-N635C Q527C-L632C_Y198C-S712CJ771 C-H802C :atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggctgtgccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctgtaggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagaccttcgacagcaccatcgcaactgagactggcaagtccatccacttttgtaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgtgtg acctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaacccc accaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgtt gcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctact cccggcccctggtgagcttctcttttatttgtgacaccaagacctacgagggccagctgggcaccgacaacgagatct tcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgt gtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgttt gatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgtgtaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaaccc cttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagac agatgagccagcagcctgtgcagatgctgtaccccggctgtgacgagctggcccagcagcacgccagcggcgag ggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgtgtgagcagaaccaggag cagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggacag gttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgagaca gagttctgataa (SEQ ID NO: 40)Construct EBVgB_mRNA_RTTR_V318C-G477C L174C-N563C S55C-N635C Q527C-L632C_Y198C-S712C:atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcctgaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggctgtgccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctgtaggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccacttttgtaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgtgtg acctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaacccc accaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgtt gcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctact cccggcccctggtgagcttctcttttatttgtgacaccaagacctacgagggccagctgggcaccgacaacgagatct tcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgt gtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgttt gatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgtgtaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaaccc cttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagac agatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcgag ggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagga gcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggaca ggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgagac agagttctgataa (SEQ ID NO: 41)Construct EB VgB_mRNA_RTTR_V318C-G477C L 174C-N563C S55C-N635C Q527C-L632C:atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggctgtgccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccacttttgtaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgtgtg acctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaacccc accaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgtt gcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctact cccggcccctggtgagcttctcttttatttgtgacaccaagacctacgagggccagctgggcaccgacaacgagatct tcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgt gtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgttt gatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacc ccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccaga cagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcgagggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagg agcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggac aggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgaga cagagttctgataa (SEQ ID NO: 42)Construct EBVgB_mRNA_RTTR_V318C-G477C S55C-N635C Q527C-L632C Y198C-S712C:atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggcctggccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctgtaggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccacttttgtaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgtgtg acctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaacccc accaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgtt gcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctact cccggcccctggtgagcttctcttttatcaacgacaccaagacctacgagggccagctgggcaccgacaacgagat cttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacg tgtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaataccagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgttt gatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgtgtaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaaccc cttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagac agatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcgag ggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagga gcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggaca ggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgagac agagttctgataa (SEQ ID NO: 43)Construct EBVgB_mRNA_RTTR_V318C-G477C S55C-N635C Q527C-L632C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggcctggccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccacttttgtaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgtgtg acctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccccaccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgtt gcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctact cccggcccctggtgagcttctcttttatcaacgacaccaagacctacgagggccagctgggcaccgacaacgagat cttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacg tgtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgttt gatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacc ccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccaga cagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcga gggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagg agcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggac aggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgaga cagagttctgataa (SEQ ID NO: 44)Construct EBVgB_mRNA_RTTR_S54C-S526C_S55C-N635C_Q527C-L632C_Y198C- S712C:atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgtgttgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagaga accacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaag atcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccgt ggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggcctggccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctgtaggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgagaagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggc gacctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccc caccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtgttgt tgcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctac tcccggcccctggtgagcttctcttttatcaacgacaccaagacctacgagggccagctgggcaccgacaacgagat cttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacg tgtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgttt gatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgtgtaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaaccc cttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagac agatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcgag ggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagga gcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggaca ggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgagac agagttctgataa (SEQ ID NO: 45)Construct EBVgB_mRNA_RTTR_S54C-S526C_S55C-N635C_Q527C-L632C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgtgttgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagaga accacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaag atcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccgt ggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggcctggccaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggccagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggc gacctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccc caccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtgttgt tgcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctac tcccggcccctggtgagcttctcttttatcaacgacaccaagacctacgagggccagctgggcaccgacaacgagat cttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacg tgtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgttt gatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacc ccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccaga cagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcga gggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagg agcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggac aggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgaga cagagttctgataa (SEQ ID NO: 46)Construct EBVgB_mRNA_RTTR_A175C-V529C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagcagccacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagaga gaaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaagatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctcc gtggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggc ctgaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggcctgtgtaac ggcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagg accaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccgg ccagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttcca cgtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcct ggacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagac cttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgaggggacctctagctttgtgac caacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacga gaagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcct gctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaac cagcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctgg gcggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggca ccctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctggg cgacctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaacc ccaccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtccc agtgctgtcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgct actcccggcccctggtgagcttctcttttatcaacgacaccaagacctacgagggccagctgggcaccgacaacga gatcttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatcc acgtgtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctg aataccagcctgatcgagaacatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaac gtgtttgatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctgg ataacgccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgg gccagtctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaa gaaccccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaa ccagacagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagc ggcgagggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaa ccaggagcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgcca gggacaggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggc cgagacagagttctgataa (SEQ ID NO: 47)Construct EBVgB_mRNA_RTTR_G322C-D478C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagcagccacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagaga gaaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacacca agatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctcc gtggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggc ctgaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggcctggccaa cggcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccag gaccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccg gccagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttcc acgtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcc tggacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcaga ccttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgagtgtacctctagctttgtgac caacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacga gaagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcct gctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaac cagcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctgg gcggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggca ccctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctggg ctgtctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccc caccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtccca gtgcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgcta ctcccggcccctggtgagcttctcttttatcaacgacaccaagacctacgagggccagctgggcaccgacaacgag atcttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatcca cgtgtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctga ataccagcctgatcgagaacatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgt gtttgatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggata acgccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggcc agtctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaa ccccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagacagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggc gagggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaacca ggagcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccaggg acaggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccga gacagagttctgataa (SEQ ID NO: 48)Construct EBVgB_mRNA_RTTR_A175C-V529C_G322C-A480C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagcagccacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagaga gaaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacacca agatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctcc gtggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggc ctgaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccaccggcggcctgtgtaac ggcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagg accaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccgg ccagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttcca cgtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcct ggacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagac cttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgagtgtacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggc gacctgtgtagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaacccc accaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcccag tgctgtcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctact cccggcccctggtgagcttctcttttatcaacgacaccaagacctacgagggccagctgggcaccgacaacgagat cttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacg tgtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaataccagcctgatcgagaacatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgtt tgatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacc ccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccaga cagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcga gggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagg agcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggac aggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgaga cagagttctgataa (SEQ ID NO: 49)Construct EBVgB_mRNA_RTTR_A175C-V529C_G172C-D564C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagcagccacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagaga gaaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacacca agatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctcc gtggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggc ctgaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccacctgtggcctgtgtaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgaggggacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctg ctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggc gacctggccagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccccaccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtccca gtgctgtcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgcta ctcccggcccctggtgagcttctcttttatcaactgtaccaagacctacgagggccagctgggcaccgacaacgaga tcttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccac gtgtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaat accagcctgatcgagaacatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgt ttgatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataa cgccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggcca gtctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaac cccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccag acagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcg agggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccag gagcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccaggga caggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgag acagagttctgataa (SEQ ID NO: 50)Construct EBVgB_mRNA_RTTR_G322C-A480C_G172C-D564C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagcagccacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagaga gaaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacacca agatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctcc gtggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggc ctgaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccacctgtggcctggccaac ggcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagg accaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccgg ccagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttcca cgtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcct ggacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagac cttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgagtgtacctctagctttgtgacc aacaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgag aagtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctgctgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacc agcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctggg cggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcac cctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggc gacctgtgtagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaacccc accaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcccag tgcgtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctac tcccggcccctggtgagcttctcttttatcaactgtaccaagacctacgagggccagctgggcaccgacaacgagat cttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacg tgtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagcctgatcgagaacatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgtt tgatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacc ccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccaga cagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcga gggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagg agcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggac aggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgaga cagagttctgataa (SEQ ID NO: 51)Construct EBVgB_mRNA_RTTR_G322C-A480C_G172C-D564C_S55C-N635C Q527C-L632C:atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccacctgtggcctggccaacgg cgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccaggac caccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggccagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccacg tgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctgg acaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacctt cgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgagtgtacctctagctttgtgaccaa caccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgagaa gtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctgct gctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaaccag cagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctgggcg gcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcaccc tgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggcga cctgtgtagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaaccccac caccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgttgc gtgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctactcc cggcccctggtgagcttctcttttatcaactgtaccaagacctacgagggccagctgggcaccgacaacgagatcttc ctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgtgt acaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaatacc agctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgtttgat ctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataacgcc gtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccagtct atcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacccc ttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagaca gatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcgagg gccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccaggag cagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggacag gttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgagaca gagttctgataa (SEQ ID NO: 52)Construct EBVgB_mRNA_RTTR_A175C-V529C_G322C-A480C_G172C-D564C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagcagccacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagaga gaaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaagatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctcc gtggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggc ctgaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccacctgtggcctgtgtaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgagtgtacctctagctttgtgacca acaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgaga agtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctgc tgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacca gcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctgggc ggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcacc ctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggcg acctgtgtagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaacccca ccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcccagt gctgtcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctact cccggcccctggtgagcttctcttttatcaactgtaccaagacctacgagggccagctgggcaccgacaacgagatc ttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgt gtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagcctgatcgagaacatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgtt tgatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacc ccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccaga cagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcga gggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagg agcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggac aggttccccggcctgagaacgacgcggtaccacgaccccgaaaccgccgccgccctgctgggcgaggccgaga cagagttctgataa (SEQ ID NO: 53)Construct EBVgB_mRNA_RRRR_A175C-V529C_G322C-A480C_G172C-D564C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagcagccacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagaga gaaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacacca agatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctcc gtggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggc ctgaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccacctgtggcctgtgtaacg gcgtgaggcggtatgcctcccagaccgagctgtacgacgcccccggctggctgatctggacctataggaccagga ccaccgtgaactgcctgatcaccgacatgatggccaagagcaacagccccttcgatttcttcgtgaccaccaccggc cagaccgtggagatgagccccttctacgacggcaagaataaggagacattccacgagagagccgacagcttccac gtgcgcaccaattacaagatcgtggactatgacaatcggggcaccaacccccagggcgagaggagagccttcctg gacaagggcacctacaccctgtcctggaagctggagaaccggaccgcctattgccccctgcagcactggcagacc ttcgacagcaccatcgcaactgagactggcaagtccatccactttgtgaccgacgagtgtacctctagctttgtgacca acaccaccgtggggatcgagctgcccgacgccttcaagtgtatcgaggaacaggtgaataagaccatgcacgaga agtacgaggccgtgcaggacaggtacacaaagggccaggaagccattacctacttcatcaccagcggcggcctgc tgctggcctggctgccactgacccccagatccctggccaccgtgaagaacctgaccgagctgaccaccccaacca gcagcccccctagcagccccagccctcctgccccctccgccgcccggggctccacccctgccgccgtgctgggc ggcgggggcagcggaagcggcaacgccaccacccccgtgccccccaccgcccccggcaaaagcctgggcacc ctgaacaaccccgccaccgtgcagatccagttcgcctacgattccctgaggagacagatcaatagaatgctgggcg acctgtgtagggcctggtgcctggagcagaagaggcagaatatggtgctgcgcgagctgaccaagatcaacccca ccaccgtgatgagcagcatttacggcaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcccagt gctgtcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctact cccggcccctggtgagcttctcttttatcaactgtaccaagacctacgagggccagctgggcaccgacaacgagatc ttcctgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgt gtacaacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaata ccagcctgatcgagaacatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgtt tgatctggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataac gccgtgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggactccctgggcagcgtgggccag tctatcacaaacctggtgagcaccgtgggcggcctgttttccagcctggtgagcggattcatcagcttcttcaagaacc ccttcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatcagtctgacccgcagaaccagacagatgagccagcagcctgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgccagcggcga gggccccggcatcaaccctatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccagg agcagaagagagccgcccagagggccgccgggcccagcgtggccagccgcgccctgcaggccgccagggac aggttccccggcctgagacggaggagataccacgaccccgaaaccgccgccgccctgctgggcgaggccgaga cagagttctgataa (SEQ ID NO: 54)Construct EBVgB_mRNA_S55C-N635C_Q527C-L632C: atgactagacggcgggtgctgagcgtggtggtgctgctggccgccctggcctgcagactgggcgcccagacccct gagcagcccgcccctcccgccaccaccgtgcagcctaccgccacaaggcagcagaccagcttccccttcagagtg tgcgagctgagctgtcacggggacctgtttaggttcagctccgacatccagtgtccttctttcgggaccagggaaaac cacactgagggcctgctgatggtgttcaaggacaacattatcccctactccttcaaggtgagaagctacaccaagatc gtgaccaacatcctgatctataacggctggtacgccgactccgtgaccaacagacacgaggagaagttcagcgtgg actcatacgaaaccgaccagatggacaccatctatcagtgctacaacgccgtgaagatgacaaaggatggactgac ccgcgtgtacgtggacagagatggcgtgaacatcaccgtgaatctgaagcccaccggcggcctggccaacggcgt gaggcggtacgcctcccagacagagctgtacgacgcccccggctggctgatctggacctacaggaccaggacca ccgtgaactgcctgatcaccgacatgatggccaagtctaacagccccttcgactttttcgtgaccaccaccgggcaga ccgtggagatgagccctttctacgacggcaagaacaaggagacattccacgagagggccgacagctttcacgtgc ggaccaattataagatcgtggactacgacaaccgggggactaacccacagggggagaggcgggccttcctggac aagggcacctacacactgtcctggaagctggaaaataggaccgcctactgtccactgcagcactggcagaccttcg acagcaccatcgccaccgaaaccggcaagtccatccacttcgtgacagatgagggcacctctagctttgtcaccaat acaaccgtgggcatcgagctgcccgatgcctttaagtgcatcgaggaacaggtgaataagaccatgcacgagaagt acgaggccgtgcaggacaggtacaccaagggccaggaggccatcacctactttatcacaagcggaggcctgctgc tggcctggctgcctctgacccccaggagcctggctaccgtgaagaacctgaccgagctgaccacccctaccagtag cccccctagttcacccagccctccagccccaagcgccgccagaggcagcacccctgcagccgtgctggggggcg gcggaagcggcagcggcaacgccacaactcccgtgccccccaccgcccccggcaagagcctgggcaccctgaa caaccccgccaccgtgcagatccagttcgcctacgacagcctgagacgccagatcaataggatgctgggcgacct ggccagggcctggtgcctggagcagaagagacagaacatggtgctgagagagctgacaaagatcaaccccacca cagtgatgagcagcatctacggcaaggccgtggccgccaagcgcctgggcgacgtgatttccgtgtcctgttgcgt gcccgtgaaccaggccaccgtgaccctgcgcaagagcatgcgggtgcccggcagcgagactatgtgctactcacg gccactggtgagcttctcttttattaacgacaccaagacatacgagggccagctgggcaccgacaatgagatctttctg accaagaagatgaccgaggtgtgccaggccaccagccagtactacttccagagcggcaatgagatccacgtgtac aacgactatcaccacttcaagaccatcgagctggacggaatcgccaccctgcagaccttcatcagcctgaacaccagctgtatcgagtgtatcgacttcgccagcctggagctgtacagcagagacgaacagagggccagcaatgtgttcgacc tggagggcatcttcagggagtacaacttccaggcccagaatatcgccggcctgagaaaggacctggacaatgccgt gtccaacggcagaaaccagtttgtggacggcctgggcgagctgatggactccctgggcagcgtgggccagagcat caccaatctggtgagcaccgtgggcggcctgttctccagcctggtgagcggcttcatcagcttcttcaagaacccatt cggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatctctctgaccaggcggaccagacag atgagccagcagcccgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgcctccggcgaggg acctggcatcaaccccatctccaagacagagctgcaggccatcatgctggccctgcacgagcagaatcaggagca gaagcgggccgctcagagagctgccggccccagtgtggcctctcgggccctgcaggccgccagagacagattcc ccggcctgaggcgccgcaggtaccacgaccccgaaaccgccgccgctctgctgggcgaggccgaaaccgagttt tgataa (SEQ ID NO: 55)Construct EBVgB_mRNA_RTTR_S55C-N635C_Q527C-L632C: atgactagacggcgggtgctgagcgtggtggtgctgctggccgccctggcctgcagactgggcgcccagacccct gagcagcccgcccctcccgccaccaccgtgcagcctaccgccacaaggcagcagaccagcttccccttcagagtg tgcgagctgagctgtcacggggacctgtttaggttcagctccgacatccagtgtccttctttcgggaccagggaaaac cacactgagggcctgctgatggtgttcaaggacaacattatcccctactccttcaaggtgagaagctacaccaagatc gtgaccaacatcctgatctataacggctggtacgccgactccgtgaccaacagacacgaggagaagttcagcgtgg actcatacgaaaccgaccagatggacaccatctatcagtgctacaacgccgtgaagatgacaaaggatggactgac ccgcgtgtacgtggacagagatggcgtgaacatcaccgtgaatctgaagcccaccggcggcctggccaacggcgt gaggcggtacgcctcccagacagagctgtacgacgcccccggctggctgatctggacctacaggaccaggacca ccgtgaactgcctgatcaccgacatgatggccaagtctaacagccccttcgactttttcgtgaccaccaccgggcaga ccgtggagatgagccctttctacgacggcaagaacaaggagacattccacgagagggccgacagctttcacgtgc ggaccaattataagatcgtggactacgacaaccgggggactaacccacagggggagaggcgggccttcctggac aagggcacctacacactgtcctggaagctggaaaataggaccgcctactgtccactgcagcactggcagaccttcg acagcaccatcgccaccgaaaccggcaagtccatccacttcgtgacagatgagggcacctctagctttgtcaccaat acaaccgtgggcatcgagctgcccgatgcctttaagtgcatcgaggaacaggtgaataagaccatgcacgagaagt acgaggccgtgcaggacaggtacaccaagggccaggaggccatcacctactttatcacaagcggaggcctgctgc tggcctggctgcctctgacccccaggagcctggctaccgtgaagaacctgaccgagctgaccacccctaccagtag cccccctagttcacccagccctccagccccaagcgccgccagaggcagcacccctgcagccgtgctggggggcg gcggaagcggcagcggcaacgccacaactcccgtgccccccaccgcccccggcaagagcctgggcaccctgaa caaccccgccaccgtgcagatccagttcgcctacgacagcctgagacgccagatcaataggatgctgggcgacct ggccagggcctggtgcctggagcagaagagacagaacatggtgctgagagagctgacaaagatcaaccccaccacagtgatgagcagcatctacggcaaggccgtggccgccaagcgcctgggcgacgtgatttccgtgtcctgttgcgt gcccgtgaaccaggccaccgtgaccctgcgcaagagcatgcgggtgcccggcagcgagactatgtgctactcacg gccactggtgagcttctcttttattaacgacaccaagacatacgagggccagctgggcaccgacaatgagatctttctg accaagaagatgaccgaggtgtgccaggccaccagccagtactacttccagagcggcaatgagatccacgtgtac aacgactatcaccacttcaagaccatcgagctggacggaatcgccaccctgcagaccttcatcagcctgaacaccag ctgtatcgagtgtatcgacttcgccagcctggagctgtacagcagagacgaacagagggccagcaatgtgttcgacc tggagggcatcttcagggagtacaacttccaggcccagaatatcgccggcctgagaaaggacctggacaatgccgt gtccaacggcagaaaccagtttgtggacggcctgggcgagctgatggactccctgggcagcgtgggccagagcat caccaatctggtgagcaccgtgggcggcctgttctccagcctggtgagcggcttcatcagcttcttcaagaacccatt cggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgatctctctgaccaggcggaccagacag atgagccagcagcccgtgcagatgctgtaccccggcatcgacgagctggcccagcagcacgcctccggcgaggg acctggcatcaaccccatctccaagacagagctgcaggccatcatgctggccctgcacgagcagaatcaggagca gaagcgggccgctcagagagctgccggccccagtgtggcctctcgggccctgcaggccgccagagacagattcc ccggcctgaggacgacgaggtaccacgaccccgaaaccgccgccgctctgctgggcgaggccgaaaccgagttt tgataa (SEQ ID NO: 56)Construct EBVgB_mRNA_RTTR_G172C-D564C_S55C-N635C_Q527C-L632C: atgacacggcggagggtgctgagtgtggtggtgctgctggccgccctggcctgcagactgggcgcccagaccccc gagcagcccgccccacccgccaccaccgtgcagcccaccgccacaaggcagcagaccagcttcccattccgggt gtgcgagctgagctgtcacggcgacctgttcaggttcagctccgacatccagtgccccagcttcggcaccagagag aaccacaccgagggcctgctgatggtgtttaaggacaacatcatcccatacagcttcaaggtgaggtcctacaccaa gatcgtgaccaacatcctgatttacaacggctggtacgctgatagcgtgaccaacaggcacgaggagaagttctccg tggatagctacgagacagaccagatggacaccatctaccagtgctacaacgccgtgaagatgaccaaagacggcct gaccagggtgtacgtggacagggacggcgtgaacatcaccgtgaatctgaagcccacctgtggcctggccaacgg cgtgaggaggtacgccagccagaccgagctgtacgatgcccccggctggctgatctggacatacaggaccagga caaccgtgaattgtctgatcaccgacatgatggccaagtccaacagccccttcgacttcttcgtgaccacaaccggcc agaccgtggagatgagccctttttacgacggcaagaacaaggaaacctttcacgagagagccgatagcttccacgt gaggaccaactacaagatcgtggactacgacaacagaggcaccaacccccagggcgagcggcgggccttcctgg ataagggcacttacaccctgagctggaagctggagaacaggaccgcctactgtcctctgcagcactggcagaccttc gactccaccatcgccaccgagactggcaagtctatccacttcgtgaccgacgagggcacaagcagcttcgtgacca acacaaccgtgggcattgagctgcccgacgccttcaagtgcatcgaggaacaggtgaacaagaccatgcacgaga agtatgaggccgtgcaggacagatacaccaagggccaggaggccatcacctactttatcaccagcggggggctgctgctggcctggctgcctctgaccccccgcagcctggccaccgtgaagaatctgaccgagctgaccacacctactag cagccccccctccagccccagcccccccgcccccagcgccgccagaggcagcacccccgccgccgtgctgggc ggcggcggctctggcagcggcaacgccaccacccccgtgccacctaccgcccccggcaagtccctgggcaccct gaacaatcccgccaccgtgcagatccagttcgcctacgatagcctgagaaggcagatcaacaggatgctgggcga cctggcccgggcttggtgcctggagcagaagcgccagaacatggtgctgcgcgagctgaccaagatcaaccccac cactgtgatgtctagcatctacggaaaggccgtggccgccaagagactgggcgacgtgatcagcgtgtcctgttgcg tgcccgtgaaccaggccaccgtgaccctgcggaagtccatgagggtgccaggcagcgagacaatgtgctactccc ggcccctggtgagcttctcttttatcaactgtaccaagacctacgagggccagctgggcaccgacaacgagatcttcc tgaccaagaagatgaccgaggtgtgtcaggccaccagccagtactatttccagagcggcaatgagatccacgtgta caacgactaccaccactttaagaccatcgagctggacggcatcgccaccctgcagaccttcatcagcctgaatacca gctgtatcgagtgtatcgacttcgcctctctggagctgtacagcagagacgagcagcgcgccagcaacgtgtttgatc tggagggcatcttcagggagtacaacttccaggcccagaacatcgccggcctgagaaaggacctggataacgccg tgtctaacggcaggaaccagttcgtggacggcctgggagagctgatggacagcctgggctccgtgggccagagca tcaccaacctggtgagcactgtgggagggctgttcagcagcctggtgagcggcttcatcagcttcttcaagaacccct tcggcggcatgctgatcctggtgctggtggccggcgtggtgatcctggtgattagcctgaccagaagaaccaggca gatgagccagcagcccgtgcagatgctgtaccccggcatcgacgagctggcccagcagcatgccagcggcgagg gccccggcatcaaccccatcagcaagaccgagctgcaggccatcatgctggccctgcacgagcagaaccaggag cagaagagagccgcccagcgcgccgccggcccctccgtggccagcagagccctgcaggccgccagggatagg ttccccggcctgagaacgacgaggtaccacgaccccgagacagccgccgccctgctgggcgaggccgagacag agttctgataa (SEQ ID NO: 57)

[0136] Accordingly, in some embodiments, the artificial mRNAs encoding the variants of the EBV gB of the present disclosure comprise an nucleic acid sequence having at least about 90%, such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, including all values and subranges therebetween, sequence identity to the nucleic acid sequence of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57. In some embodiments, the artificial mRNAs encoding the variants of the EBV gB of the present disclosure comprise or consist of the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57.

[0137] RNAs

[0138] In some embodiments, the vaccine or immunogenic compositions disclosed herein may comprise one or more self-amplifying ribonucleic acids encoding a variant of the EBV gB as disclosed herein. Antigen expression from traditional mRNA is proportional to the number of mRNA molecules successfully delivered to a subject from a vaccine or immunogenic composition. Self-amplifying RNA, however, comprise genetically-engineered replicons derived from self¬ replicating viruses, and therefore may be added to a vaccine or immunogenic composition in lower dosages than traditional mRNA while achieving comparable results.

[0139] In some embodiments, the artificial RNAs (e.g., the artificial mRNAs) of the present disclosure comprise an open reading frame (ORF) encoding the variant of the EBV gB as disclosed herein. In some embodiments, the RNAs (e.g., mRNAs) further comprises at least one 5' UTR, 3' UTR, poly(A) tail, and / or 5' cap.A. 5' Cap

[0140] An mRNA 5' cap can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency. Several types of 5' caps are known. A 7-methylguanosine cap (also referred to as “m7G” or “Cap-0”) comprises a guanosine that is linked through a 5'-5'-triphosphate bond to the first transcribed nucleotide.

[0141] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Publication No. US 2016 / 0032356 and U.S. Publication No. US 2018 / 0125989, which are incorporated herein by reference.

[0142] 5' -capping of polynucleotides may be completed concomitantly during the in vitro-transcription reaction using the following chemical RNA cap analogs to generate the 5'-guanosine cap structure according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5')G (the ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5’)G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies). 5'-capping of modified RNA may be completed post-transcriptionally using a vaccinia virus capping enzyme to generate the Cap 0 structure: m7G(5')ppp(5')G. Cap 1 structure may be generated using both vaccinia virus capping enzyme and a 2'-0 methyltransferase to generate: m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-O-methylation of the 5'-antepenultimate nucleotide using a 2'-O methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-O-methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase.

[0143] In certain embodiments, the mRNA of the disclosure comprises a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (the ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG.

[0144] In certain embodiments, the mRNA of the disclosure comprises a 5' cap of:B. Untranslated Region (UTR)

[0145] In some embodiments, the mRNA of the disclosure includes a 5' and / or 3' untranslated region (UTR). In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon. The 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal.

[0146] In some embodiments, the mRNA disclosed herein may comprise a 5' UTR that includes one or more elements that affect an mRNA’s stability or translation. In some embodiments, a 5' UTR may be about 10 to 5,000 nucleotides in length. In some embodiments, a 5' UTR may be about 50 to 500 nucleotides in length. In some embodiments, the 5' UTR is at leastabout 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length, or about 5,000 nucleotides in length.

[0147] In some embodiments, the mRNA disclosed herein may comprise a 3' UTR comprising one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' UTR may be 50 to 5,000 nucleotides in length or longer. In some embodiments, a 3' UTR may be 50 to 1,000 nucleotides in length or longer. In some embodiments, the 3' UTR is at least about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length, or about 5,000 nucleotides in length.

[0148] In some embodiments, the mRNA disclosed herein may comprise a 5' or 3' UTR that is derived from a gene distinct from the one encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0149] In certain embodiments, the 5' and / or 3' UTR sequences can be derived from mRNA which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) toincrease the stability of the mRNA. For example, a 5' UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof, to improve the nuclease resistance and / or improve the half-life of the mRNA. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof, to the 3' end or untranslated region of the mRNA. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA relative to their unmodified counterparts, and include, for example, modifications made to improve such mRNA resistance to in vivo nuclease digestion.

[0150] Exemplary 5' UTRs include a sequence derived from a CMV immediate-early 1 (IE1) gene (U.S. Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference), or the sequence GGGAUCCUACC (SEQ ID NO: 30) (U.S. Publication No.2016 / 0151409, incorporated herein by reference).

[0151] In various embodiments, the 5' UTR may be derived from the 5' UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-associated translational regulation. However, TOP genes with a tissue specific translational regulation are also known. In certain embodiments, the 5' UTR derived from the 5' UTR of a TOP gene lacks the 5' TOP motif (the oligopyrimidine tract) (e.g., U.S. Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0152] In certain embodiments, the 5' UTR is derived from a ribosomal protein Large 32 (L32) gene (U.S. Publication No. 2017 / 0029847, supra).

[0153] In certain embodiments, the 5' UTR is derived from the 5' UTR of a hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (U.S. Publication No. 2016 / 0166710, supra).

[0154] In certain embodiments, the 5' UTR is derived from the 5' UTR of an ATP5A1 gene (U.S. Publication No. 2016 / 0166710, supra).

[0155] In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR.

[0156] In some embodiments, the 5' UTR comprises a nucleic acid sequence of GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACC GGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG UGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 31).

[0157] In some embodiments, the 3' UTR comprises a nucleic acid sequence of CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCC ACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 32).

[0158] The 5' UTR and 3' UTR are described in further detail in International Pub. No. WO 2012 / 075040, incorporated herein by reference.C. Poly adenylated Tail

[0159] As used herein, the terms “poly(A) sequence,” “poly(A) tail,” and “poly(A) region” refer to a sequence of adenosine nucleotides at the 3' end of the mRNA molecule. The poly(A) tail may confer stability to the mRNA and protect it from exonuclease degradation. The poly(A) tail may enhance translation. In some embodiments, the poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides may have essentially a length of 100 nucleotides. In certain embodiments, the poly(A) tail may be interrupted by at least one nucleotide different from an adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide, or a stretch of nucleotides, that are different from an adenosine nucleotide).

[0160] The “poly(A) tail,” as used herein, typically relates to RNA. However, in the context of the disclosure, the term likewise relates to corresponding sequences in a DNA molecule (e.g., a “poly(T) sequence”).

[0161] The poly(A) tail may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0162] In some embodiments where the nucleic acid is an RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during RNA in vitro transcription. In certain embodiments, the poly(A) tail is obtained in vitro by common methods of chemical synthesis without being transcribed from a DNA template. In various embodiments, poly(A) tails are generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols, or alternatively, by using immobilizedpoly(A) polymerases, e.g., using methods and means as described in International Pub. No. WO 2016 / 174271.

[0163] The nucleic acid may comprise a poly(A) tail obtained by enzymatic polyadenylation, wherein the majority of nucleic acid molecules comprise about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0164] In some embodiments, the nucleic acid may comprise a poly(A) tail derived from a template DNA and may additionally comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in International Pub. No. WO 2016 / 091391.

[0165] In certain embodiments, the nucleic acid comprises at least one polyadenylation signal.

[0166] In various embodiments, the nucleic acid may comprise at least one poly(C) sequence.

[0167] The term “poly(C) sequence,” as used herein, is intended to be a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides.D. Chemical Modification

[0168] The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may comprise at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications can include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNA may be synthesized from modified nucleotide analogues or derivatives of purines and pyrimidines, such as, e.g., 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxy acetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5’ -methoxy carbonylmethyl-uracil, 5-methoxy -uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, P-D-mannosyl-queosine, phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0169] In some embodiments, the disclosed mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1 -methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-m ethyl- 1-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-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-m ethyluridine, 5 -methoxyuridine, and 2'-O-methyl uridine.

[0170] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof.

[0171] In some embodiments, the chemical modification comprises N1 -methylpseudouridine. Typically, the chemical modification comprises N1 -methylpseudouridine in place of every uridine, i.e. 100% of U residues are N1 -methylpseudouridine.

[0172] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, including all values and subranges therebetween, of the uracil nucleotides in the mRNA are chemically modified.

[0173] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, including all values and subranges therebetween, of the uracil nucleotides in the ORF are chemically modified.

[0174] The preparation of such analogues is described, e.g., in U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642.E. mRNA Synthesis

[0175] The mRNAs disclosed herein may be synthesized according to any of a variety of methods. For example, mRNAs according to the present disclosure may be synthesized via in vitro transcription (IVT). Some methods for in vitro transcription are described, e.g., in Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14. Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions may vary according to the specific application. The presence of these reagents is generally undesirable in a final mRNA product and these reagents can be considered impurities or contaminants which can be purified or removed to provide a clean and / or homogeneous mRNA that is suitable for therapeutic use. While mRNA provided from in vitro transcription reactions may be desirable in some embodiments, other sources of mRNA can be used according to the instant disclosure including wild-type mRNA produced from bacteria, fungi, plants, and / or animals.Immunogenic Compositions and Vaccines

[0176] The present disclosure also relates to immunogenic compositions that comprise any of the variants of the EBV gB disclosed herein or any of the artificial nucleic acids or vectors encoding such variants of the EBV gB. As used herein, the term “immunogenic composition” refers to a composition that generates an immune response that may or may not be a protective immune response or protective immunity. The term “immune response” refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen, immunogen, or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate and / or adaptive immune response. Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and / or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production and the like. An antibody response or humoral response is an immune response in which antibodies are produced. A “cellular immune response” is one mediated by T cells and / or other white blood cells. The immunogenic compositions described herein may elicitantibodies (e.g. neutralizing antibodies) and / or a T cell response against EBV gB, for example antibodies (e.g. neutralizing antibodies.

[0177] Also provided herein is a vaccine comprising the immunogenic composition of the present disclosure and a pharmaceutically acceptable carrier. Also provided herein is a vaccine comprising (i) a variant of the EBV gB disclosed herein or an artificial nucleic acid (e.g., mRNA) as described herein encoding a variant of the EBV gB disclosed herein; and (ii) a pharmaceutically acceptable carrier. As used herein, the term “vaccine” refers to a composition that generates a protective immune response or protective immunity in a subject. A “protective immune response” or “protective immunity” refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection) or prevents or reduces at least one symptom of infection (for instance, an infection by EBV). Vaccines may elicit both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous, intraperitoneal, intradermal, intranasal, by inhalation, or intramuscular.

[0178] The term “pharmaceutically acceptable” means that the carrier, at the dosages and concentrations employed, will not cause unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see e.g., Remington’s Pharmaceutical Sciences, 19thed., Mack Publishing Co., Easton, PA, 1995; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis, 2000; and Handbook of Pharmaceutical Excipients, 3rded., A. Kibbe, Ed., Pharmaceutical Press, 2000). The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can, e.g., be employed as liquid carriers, particularly for injectable solutions. The exact formulation should suit the mode of administration. The variants of the EBV gB and / or nucleic acid (e.g., mRNA) molecules preferably are formulated and administered as a sterile solution. Sterile solutions are prepared by sterile filtration or by other methods known in the art. The solutions can then be lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, such as pH 5.0 to 7.5.

[0179] Accordingly, in some embodiments, provided herein is a composition comprising any of the variants of the EBV gB disclosed herein, a trimeric EBV gB complex comprising three copies of any of the variants of the EBV gB disclosed herein, an artificial nucleic acid encoding any of the variants of the EBV gB disclosed herein, a vector comprising such an artificial nucleic acid, or a VLP comprising any of the variants of the EBV gB disclosed herein. In some embodiments, provided herein is a composition comprising one or more mRNA molecules encapsulated in a lipid nanoparticle (LNP), wherein the one or more mRNA encode any of the variants of the EBV gB disclosed herein. In some embodiments, such a composition is an immunogenic composition.

[0180] In some embodiments, also provided herein is an immunogenic composition or vaccine comprising any of the variants of the EBV gB disclosed herein. In some embodiments, provided herein is an immunogenic composition or vaccine comprising a trimeric EBV gB complex comprising three copies of any of the variants of the EBV gB disclosed herein. In some embodiments, provided herein is an immunogenic composition or vaccine comprising an artificial nucleic acid molecule, or a vector comprising such an artificial nucleic acid molecule, that encodes any of the variants of the EBV gB disclosed herein. In some embodiments, provided herein is an immunogenic composition or vaccine comprising a VLP comprising any of the variants of the EBV gB disclosed herein. In some embodiments, provided herein is an immunogenic composition or vaccine comprising one or more messenger RNA (mRNA) molecules encoding any of the variants of the EBV gB disclosed herein. In certain embodiments, the one or more mRNA molecules in the immunogenic composition or vaccine of the disclosure are encapsulated in a lipid nanoparticle (LNP).

[0181] In some embodiments, the immunogenic compositions or vaccines comprise other polypeptides in addition to the variants of the EBV gB disclosed herein disclosed herein. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode, more than one polypeptide (e.g., two, three, four, five, six, seven, eight, nine, ten, or more polypeptides). In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode, two polypeptides. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode, three polypeptides.

[0182] In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the EBVglycoprotein complex gH / gL. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the EBV glycoprotein gp42. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the EBV glycoprotein gp350. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the EBV glycoprotein gp220. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the T cell antigen BZLF1. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the T cell antigen BMRF1. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the T cell antigen EBNA3a. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the T cell antigen EBNA3b. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the T cell antigen EBNA3c. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the T cell antigen LMP2. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the T cell antigen BRFL1. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the T cell antigen BMLF1. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and the T cell antigen EBNA-1. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode any of the variants of the EBV gB disclosed herein and any combination of the EBV glycoproteins gH / gL, gp42, gp350, and / or gp220, and / or T cell antigens BZLF1, BMRF1, EBNA3a, EBNA3b, EBNA3c, LMP2, BRFL1, BMLF1, and / or EBNA-1.

[0183] Each ribonucleic acid molecule may be present in the compositions disclosed herein in an amount effective to induce an immune response in a subject to which the composition isadministered. In certain embodiments, each ribonucleic acid molecule may be present in the vaccine or immunogenic compositions disclosed herein in an amount ranging, for example, from about 0.1 pg to about 150 pg, such as from about 5 pg to about 120 pg, from about 10 pg to about 60 pg, or about 15 pg to about 45 pg, including all values and subranges therebetween. In certain embodiments, each ribonucleic acid molecule is present in the vaccine or immunogenic composition in an amount sufficient to encode, for example, from about 5 pg to about 120 pg, such as from about 10 pg to about 60 pg, or about 15 pg to about 45 pg, including all values and subranges therebetween, of the variants of the EBV gB disclosed herein.

[0184] The LNP compositions of the present disclosure may be provided as a frozen liquid form or a lyophilized form. A variety of cryoprotectants may be used, including, without limitations, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant may constitute 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition comprises trehalose, e.g., at 5-30% (e.g., 10%) (w / v). Once formulated with the cryoprotectant, the LNP compositions may be frozen (or lyophilized and cryopreserved) at -20°C to -80°C. The LNP compositions may be provided to a patient in an aqueous buffered solution -thawed if previously frozen, or if previously lyophilized, reconstituted in an aqueous buffered solution at bedside. The buffered solution preferably is isotonic and suitable for e.g., intramuscular or intradermal injection. In some embodiments, the buffered solution is a phosphate-buffered saline (PBS).

[0185] In some embodiments, the composition of the disclosure is an immunogenic composition capable of eliciting an immune response (e.g., an antibody response such as neutralizing antibodies) against EBV in a subject.

[0186] In some embodiments, the immunogenic composition or vaccine of the disclosure may further comprise one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients to stabilize the variants of the EBV gB comprised therein, or mRNA molecules encoding the same and / or LNP encapsulating such mRNA molecules, or to facilitate administration of the immunogenic composition or vaccine. Examples of such excipients include, but are not limited to, parabens, thimerosal, thiomersal, chlorobutanol, bezalkonium chloride, and chelators (e.g., ethylenediaminetetraacetic acid, or EDTA).I. Lipid Nanoparticle

[0187] The term “lipid nanoparticle” or “LNP” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids, for example a cationic lipid and / or non-cationic lipid, and one or more excipients selected from neutral lipids, anionic lipids, zwitterionic lipids, ionizable lipids, steroids, and polymer conjugated lipids (e.g., a pegylated lipid). Examples of suitable lipids include, but are not limited to, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). RNA-encapsulated LNP compositions are known in the art, such as those described in PCT Publication Nos. WO 2021 / 237084 and WO 2022 / 099003, the entire contents of which are incorporated by reference herein.

[0188] Any known LNP formulations may be used in the embodiments disclosed herein. In some embodiments, the LNPs comprise four categories of lipids: (i) an ionizable lipid (e.g., a cationic lipid); (ii) a PEGylated lipid; (iii) a cholesterol -based lipid, and (iv) a helper lipid.A. Cationic Lipid

[0189] An ionizable lipid facilitates mRNA encapsulation and may be a cationic lipid. A cationic lipid affords a positively charged environment at low pH to facilitate efficient encapsulation of the negatively charged mRNA drug substance. Exemplary cationic lipids are shown below in Table 3.Table 3. Cationic lipids.><< ><>">

[0190] The cationic lipid may be selected from the group comprising [ckkE10] / [OF-02], [(6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl]4-(dimethy < la mino)butanoate (D-Lin-MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA); 1,2-dilinoleyloxy-N,N-dimethyl-3 -aminopropane (DLin-DMA); di((Z)-non-2-en-l-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); [(4-hydroxybutyl)azanediyl]di(hexane-6, l-diyl)bis(2-hexyl decanoate) (ALC-0315); [3-(dimethylamino)-2-[(Z)-octadec-9-enoyl] oxypropyl](Z)-octadec-9-enoate (DODAP); 2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl) amino]-2-oxoethyl]pentanamide (DOGS); [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,l l,12,14,15,16,17-dodecahydro-lH-cyclopenta[a] phenanthren-3-yl]N-[2-(dimethylamino)ethyl]carbamate (DC-Chol); tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl)bis(propane-3,ldiyl))bis(azanetriyl))tetrapropionate (3060il0); decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9); ethyl 5,5-di((Z)-heptadec-8-en-l-yl)-l-(3-(pyrrolidin-l-yl)propyl)-2,5-dihydro-lH-imidazole-2-carboxylate (A2-Iso5-2DC18); bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl) azanediyl)dipropionate (B AME-016B); 1 , 1 '-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (Cl 2-200); 3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (cKK-E12); hexa(octan-3-yl) 9,9',9'',9"',9'''',9"'"-((((benzene-l,3,5-tricarbonyl)yris(azanediyl))tris(propane-3,l-diyl))tris (azanetriyl))hexanonanoate (FTT5); (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,l-diyl))bis (azanetriyl))tetrakis(ethane-2, l-diyl)(9Z,9'Z,9"Z,9"'Z, 12Z, 12'Z, 12"Z, 12"'Z)-tetrakis(octadeca-9, 12-di enoate) (OF-Deg-Lin); TT3; Nl,N3,N5-tris(3-(didodecylamino)propyl)benzene-l,3,5-tricarboxamide; Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino] butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5); IM-001; and combinations thereof.

[0191] In certain embodiments, the cationic lipid is biodegradable. In various embodiments, the cationic lipid is not biodegradable. In some embodiments, the cationic lipid is cleavable. In certain embodiments, the cationic lipid is not cleavable.

[0192] Cationic lipids are described in further detail in Dong et al. (PNAS. 111(11):3955-60.2014); Fenton et al. (Adv. Mater. 28:2939. 2016); U.S. Pat. No. 9,512,073; and U.S. Pat. No.10,201,618, each of which is incorporated herein by reference.B. PEGylated Lipid

[0193] The PEGylated lipid component provides control over particle size and stability of the nanoparticle. The addition of such components may prevent complex aggregation and provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid pharmaceutical composition to target tissues (Klibanov et al., FEBS Letters 268(l):235-7. 1990). These components may be selected to rapidly exchange out of the pharmaceutical composition in vivo (see, e.g., U.S. Pat. No. 5,885,613).

[0194] Contemplated PEGylated lipids include, but are not limited to, a polyethylene glycol (PEG) chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 (e.g., C8, CIO, C12, C14, C16, or C18) length, such as a derivatized ceramide (e.g., N-octanoyl-sphingosine-l-[succinyl(m ethoxypoly ethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipid is l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol (DMG-PEG); l,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol (DSPE-PEG); l,2-dilauroyl-sn-glycero-3 -phosphoethanolamine-poly ethylene glycol (DLPE-PEG); or 1,2-distearoyl-rac-glycero-polyethelene glycol (DSG-PEG), PEG-DAG; PEG-PE; PEG-S-DAG;PEG-S-DMG; PEG-cer; a PEG-dialkyoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecyl acetamide (ALC-0159); and combinations thereof.

[0195] In certain embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In certain embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159 (2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000.C. Cholesterol-Based Lipid

[0196] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNPs comprise one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example: DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Pat.5,744,335), imidazole cholesterol ester (“ICE”; WO2011 / 068810), sitosterol (22,23-dihydrostigmasterol), P-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol; desmosterol (3B-hydroxy-5,24-cholestadiene); lanosterol (8,24-lanostadien-3b-ol); 7-dehydrocholesterol (A5,7-cholesterol); dihydrolanosterol (24,25 -dihydrolanosterol); zymosterol (5a-cholesta-8,24-dien-3B-ol); lathosterol (5a-cholest-7-en-3B-ol); diosgenin ((3p,25R)-spirost-5-en-3-ol); campesterol (campest-5-en-3B-ol); campestanol (5a-campestan-3b-ol); 24-methylene cholesterol (5,24(28)-cholestadien-24-methylen-3B-ol); cholesteryl margarate (cholest-5-en-3B-yl heptadecanoate); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNPs is cholesterol.D. Helper Lipid

[0197] A helper lipid enhances the structural stability of the LNP and helps the LNP in endosome escape. It improves uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is a zwitterionic lipid, which has fusogenic properties for enhancing uptake and release of the drug payload. Examples of helper lipids are l,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE); 1,2-di stearoyl -sn-glycero-3 -phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); l,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE); and l,2-dioleoyl-sn-glycero-3 -phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, l,2-dilauroyl-sn-glycero-3 -phosphocholine(DLPC), 1,2-Distearoylphosphatidyl ethanolamine (DSPE), and l,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0198] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelins, ceramides, cerebrosides, gangliosides, 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a combination thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0199] In various embodiments, the present LNPs comprise (i) a cationic lipid selected from OF-02, cKK-ElO, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.E. Molar Ratios of the Lipid Components

[0200] The molar ratios of the herein components are important for the LNPs’ effectiveness in delivering mRNA. The molar ratio of the cationic lipid, the PEGylated lipid, the cholesterol-based lipid, and the helper lipid is A: B: C: D, where A + B + C + D = 100%. In some embodiments, the molar ratio of the cationic lipid in the LNPs relative to the total lipids (i.e., A) is 35-55%, such as 35-50% (e.g., 38-42% such as 40%, or 45-50%). In some embodiments, the molar ratio of the PEGylated lipid component relative to the total lipids (i.e., B) is 0.25-2.75% (e.g., 1-2% such as 1.5%). In some embodiments, the molar ratio of the cholesterol-based lipid relative to the total lipids (i.e., C) is 20-50% (e.g., 27-30% such as 28.5%, or 38-43%). In some embodiments, the molar ratio of the helper lipid relative to the total lipids (i.e., D) is 5-35% (e.g., 28-32% such as 30%, or 8-12%, such as 10%). In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs contain a molar ratio of the cationic lipid to the helper lipid that is more than 1.

[0201] In certain embodiments, the LNP of the disclosure comprises:i) a cationic lipid at a molar ratio of 35% to 55% or 40% to 50% (e.g., a cationic lipid at a molar ratio of 35%, 36%, 37%, 38%, 39%, 40%, 41% 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%);ii) a polyethylene glycol (PEG) conjugated (PEGylated) lipid at a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., a PEGylated lipid at a molar ratio of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%);iii) a cholesterol-based lipid at a molar ratio of 20% to 45%, 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., a cholesterol-based lipid at a molar ratio of 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%, or 50%); andiv) a helper lipid at a molar ratio of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., a helper lipid at a molar ratio of 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%, or 35%),wherein all of the molar ratios are relative to the total lipid content of the LNP.

[0202] In certain embodiments, the LNP comprises: a cationic lipid at a molar ratio of 40%; a PEGylated lipid at a molar ratio of 1.5%; a cholesterol-based lipid at a molar ratio of 28.5%; and a helper lipid at a molar ratio of 30%.

[0203] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

[0204] In various embodiments, the cholesterol-based lipid is cholesterol.

[0205] In some embodiments, the helper lipid is l,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

[0206] In certain embodiments, the LNP comprises: OF-02 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0207] In certain embodiments, the LNP comprises: cKK-ElO at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0208] In certain embodiments, the LNP comprises: GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0209] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0210] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0211] In certain embodiments, the LNP comprises: SM-102 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0212] In certain embodiments, the LNP comprises: ALC-0315 at a molar ratio of 35% to 55%; ALC-0159 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0213] In certain embodiments, the LNP comprises: OF-02 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0214] In certain embodiments, the LNP comprises: cKK-ElO at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0215] In certain embodiments, the LNP comprises: GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0216] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0217] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0218] In certain embodiments, the LNP comprises: 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) at a molar ratio of 50%; 1,2-distearoyl- w-glycero-3 -phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 38.5%;and l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG2000) at a molar ratio of 1.5%.

[0219] In certain embodiments, the LNP comprises: (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 46.3%; 1,2-distearoyl-sw-glycero-3 -phosphocholine (DSPC) at a molar ratio of 9.4%; cholesterol at a molar ratio of 42.7%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a molar ratio of 1.6%.

[0220] In certain embodiments, the LNP comprises: (4-hydroxybutyl)azanediyl]di(hexane-6,1 -diyl) bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 47.4%; l,2-distearoyl-.s / / -glycero-3 -phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 40.9%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a molar ratio of 1.7%.

[0221] In certain embodiments, the LNP comprises: IM-001 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0222] To calculate the actual amount of each lipid to be put into an LNP formulation, the molar amount of the cationic lipid is first determined based on a desired N / P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP. Next, the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the molar ratio selected. These molar amounts are then converted to weights using the molecular weight of each lipid.

[0223] Accordingly, in some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding any of the variants of the EBV gB disclosed herein encapsulated in a LNP, wherein the LNP comprises a cationic lipid. In some embodiments, the cationic lipid comprises or is OF-02. In some embodiments, the cationic lipid comprises or is cKK-ElO. In some embodiments, the cationic lipid comprises or is GL-HEPES-E3-E10-DS-3-E18-1. In some embodiments, the cationic lipid comprises or is GL-HEPES-E3-E12-DS-4-E10. In some embodiments, the cationic lipid comprises or is GL-HEPES-E3-E12-DS-3-E14. In some embodiments, the cationic lipid comprises or is (4-hydroxybutyl)azanediyl] di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315). In some embodiments, the cationic lipid comprises or is IM-001.

[0224] In some embodiments, the LNP encapsulating the artificial mRNA of the present disclosure further comprises a PEGylated lipid, a cholesterol-based lipid, and a helper lipid. Insome embodiments, the PEGylated lipid comprises or is DMG-PEG2000. In some embodiments, the cholesterol-based lipid comprises or is cholesterol. In some embodiments, the helper lipid comprises or is DOPE. In some embodiments, the LNP comprises the cationic lipid at a molar ratio between about 35% and about 55%, the PEGylated lipid at a molar ratio between about 0.25% and about 2.75%, the cholesterol -based lipid at a molar ratio between about 20% and about 45%, and the helper lipid at a molar ratio between about 5% and about 35%, wherein all of the molar ratios are relative to the total lipid content of the LNP. In some embodiments, the LNP comprises the cationic lipid at a molar ratio of about 40%, the PEGylated lipid at a molar ratio of about 1.5%, the cholesterol-based lipid at a molar ratio of about 28.5%, and the helper lipid at a molar ratio of about 30%, wherein all of the molar ratios are relative to the total lipid content of the LNP.

[0225] In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding the variants of the EBV gB of SEQ ID NO: 20 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding the variants of the EBV gB of SEQ ID NO: 21 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding the variants of the EBV gB of SEQ ID NO: 22 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding the variants of the EBV gB of SEQ ID NO: 23 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding the variants of the EBV gB of SEQ ID NO: 24 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding the variants of the EBV gB of SEQ ID NO: 25 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding the variants of the EBV gB of SEQ ID NO: 26 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding the variants of the EBV gB of SEQ ID NO: 27 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%.

[0226] In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 33 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 34 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 35 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 36 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at amolar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 37 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 38 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 39 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 40 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 41 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 42 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 43 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In someembodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 44 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 45 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 46 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 47 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 48 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 49 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 50 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleicacid sequence of SEQ ID NO: 51 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 52 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 53 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 54 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 55 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 56 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 57 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%.IL Processes for Making LNP Vaccines

[0227] The LNPs can be prepared by various techniques presently known in the art. For example, multilamellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion that results in the formation of MLVs. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0228] Various methods are described in Patent Application Pub. Nos. US 2011 / 0244026, US 2016 / 0038432, US 2018 / 0153822, US 2018 / 0125989, and US 2021 / 0046192 and can be used for making LNP vaccines. One exemplary process entails encapsulating mRNA by mixing it with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles, as described in Patent Application Pub. No. US 2016 / 0038432. Another exemplary process entails encapsulating mRNA by mixing pre-formed LNPs with mRNA, as described in Patent Application Pub. No. US 2018 / 0153822.

[0229] In some embodiments, the process of preparing mRNA-loaded LNPs includes a step of heating one or more of the solutions to a temperature greater than ambient temperature, the one or more solutions being the solution comprising the pre-formed lipid nanoparticles, the solution comprising the mRNA and the mixed solution comprising the LNP-encapsulated mRNA. In some embodiments, the process includes the step of heating one or both of the mRNA solution and the pre-formed LNP solution, prior to the mixing step. In some embodiments, the process includes heating one or more of the solutions comprising the pre-formed LNPs, the solution comprising the mRNA and the solution comprising the LNP-encapsulated mRNA, during the mixing step. In some embodiments, the process includes the step of heating the LNP-encapsulated mRNA, after the mixing step. In some embodiments, the temperature to which one or more of the solutions is heated is or is greater than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C, including all values and subranges therebetween. In some embodiments, the temperature to which one or more of the solutions is heated ranges from about 25-70°C, about 30-70°C, about 35-70°C, about 40-70°C, about 45-70°C, about 50-70°C, or about 60-70°C, including all values and subranges therebetween. In some embodiments, the temperature is about 65°C.Ill

[0230] Various methods may be used to prepare an mRNA solution suitable for the present invention. In some embodiments, mRNA may be directly dissolved in a buffer solution described herein. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water or a buffer at a concentration at or greater than about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml, including all values and subranges therebetween.

[0231] In some embodiments, an mRNA stock solution is mixed with a buffer solution using a pump. Exemplary pumps include but are not limited to gear pumps, peristaltic pumps and centrifugal pumps. Typically, the buffer solution is mixed at a rate greater than that of the mRNA stock solution. For example, the buffer solution may be mixed at a rate at least lx, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 15x, or 20x greater than the rate of the mRNA stock solution. In some embodiments, a buffer solution is mixed at a flow rate ranging between about 100-6000 ml / minute (e.g., about 100-300 ml / minute, 300-600 ml / minute, 600-1200 ml / minute, 1200-2400 ml / minute, 2400-3600 ml / minute, 3600-4800 ml / minute, 4800-6000 ml / minute, or 60-420 ml / minute, including all values and subranges therebetween). In some embodiments, a buffer solution is mixed at a flow rate of, or greater than, about 60 ml / minute, 100 ml / minute, 140 ml / minute, 180 ml / minute, 220 ml / minute, 260 ml / minute, 300 ml / minute, 340 ml / minute, 380 ml / minute, 420 ml / minute, 480 ml / minute, 540 ml / minute, 600 ml / minute, 1200 ml / minute, 2400 ml / minute, 3600 ml / minute, 4800 ml / minute, or 6000 ml / minute, including all values and subranges therebetween.

[0232] In some embodiments, an mRNA stock solution is mixed at a flow rate ranging between about 10-600 ml / minute (e.g., about 5-50 ml / minute, about 10-30 ml / minute, about 30-60 ml / minute, about 60-120 ml / minute, about 120-240 ml / minute, about 240-360 ml / minute, about 360-480 ml / minute, or about 480-600 ml / minute, including all values and subranges therebetween). In some embodiments, an mRNA stock solution is mixed at a flow rate of or greater than about 5 ml / minute, 10 ml / minute, 15 ml / minute, 20 ml / minute, 25 ml / minute, 30 ml / minute, 35 ml / minute, 40 ml / minute, 45 ml / minute, 50 ml / minute, 60 ml / minute, 80 ml / minute, 100ml / minute, 200 ml / minute, 300 ml / minute, 400 ml / minute, 500 ml / minute, or 600 ml / minute, including all values and subranges therebetween.

[0233] The process of incorporation of a desired mRNA into a lipid nanoparticle is referred to as “loading.” Exemplary methods are described in Lasic et al., FEBSLet. (1992) 312:255-8. The LNP-incorporated nucleic acids may be completely or partially located in the interior space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the exterior surface of the lipid nanoparticle membrane. The incorporation of an mRNA into lipid nanoparticles is also referred to herein as “encapsulation” wherein the nucleic acid is entirely or substantially contained within the interior space of the lipid nanoparticle.

[0234] Suitable LNPs may be made in various sizes. In some embodiments, decreased size of lipid nanoparticles is associated with more efficient delivery of an mRNA. Selection of an appropriate LNP size may take into consideration the site of the target cell or tissue and to some extent the application for which the lipid nanoparticle is being made.

[0235] A variety of methods known in the art are available for sizing of a population of lipid nanoparticles. Preferred methods herein utilize Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 pl of an LNP sample are mixed with 990 pl of 10% trehalose. This solution is loaded into a cuvette and then put into the Zetasizer machine. The z-average diameter (nm), or cumulants mean, is regarded as the average size for the LNPs in the sample. The Zetasizer machine can also be used to measure the poly dispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. Average LNP diameter may be reduced by sonication of formed LNP. Intermittent sonication cycles may be alternated with quasi-elastic light scattering (QELS) assessment to guide efficient lipid nanoparticle synthesis.

[0236] In some embodiments, the majority of purified LNPs, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, including all values and subranges therebetween, of the LNPs, have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm, including all values and subranges therebetween). In some embodiments, substantially all (e.g., greater than 80 or 90%) of the purified lipid nanoparticles have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm, including all values and subranges therebetween).

[0237] In certain embodiments, the LNP has an average diameter of 30-200 nm. In various embodiments, the LNP has an average diameter of 80-150 nm.

[0238] In some embodiments, the LNPs in the present composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm, including all values and subranges therebetween.

[0239] In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, including all values and subranges therebetween, of the LNPs in the present composition have a size ranging from about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm, including all values and subranges therebetween) or about 50-70 nm (e.g., 55-65 nm) are particular suitable for pulmonary delivery via nebulization.

[0240] In some embodiments, the dispersity, or measure of heterogeneity in size of molecules (PDI), of LNPs in a pharmaceutical composition provided by the present invention is less than about 0.5. In some embodiments, an LNP has a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08, including all values and subranges therebetween. The PDI may be measured by a Zetasizer machine as described above.

[0241] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, including all values and subranges therebetween, of the purified LNPs in a pharmaceutical composition provided herein encapsulate an mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in a pharmaceutical composition encapsulate an mRNA within each individual particle. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of 50% to 99%; or greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or 99%, including all values and subranges therebetween. Typically, lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91%, 92%, 93%, 94%, or 95%, including all values and subranges therebetween).

[0242] In some embodiments, an LNP has a N / P ratio of between 1 and 10. In some embodiments, a lipid nanoparticle has a N / P ratio above 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8, including all values and subranges therebetween. In further embodiments, a typical LNP herein has an N / P ratio of 4.

[0243] In some embodiments, a pharmaceutical composition according to the present invention contains at least about 0.5 pg, 1 pg, 5 pg, 10 pg, 100 pg, 500 pg, or 1000 pg, including all values and subranges therebetween, of encapsulated mRNA. In some embodiments, a pharmaceutical composition contains about 0.1 pg to 1000 pg, at least about 0.5 pg, at least about 0.8 pg, at least about 1 pg, at least about 5 pg, at least about 8 pg, at least about 10 pg, at least about 50 pg, at least about 100 pg, at least about 500 pg, or at least about 1000 pg, including all values and subranges therebetween, of encapsulated mRNA.

[0244] In some embodiments, mRNA can be made by chemical synthesis or by in vitro transcription (IVT) of a DNA template. For example, in an IVT process, a cDNA template is used to produce an mRNA transcript and the DNA template is degraded by a DNase. The transcript is purified by depth filtration and tangential flow filtration (TFF). The purified transcript is further modified by adding a cap and a tail, and the modified RNA is purified again by depth filtration and TFF.

[0245] The mRNA is then prepared in an aqueous buffer and mixed with an amphiphilic solution containing the lipid components of the LNPs. An amphiphilic solution for dissolving the four lipid components of the LNPs may be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer may be, for example, a citrate, phosphate, acetate, or succinate buffer and may have a pH of about 3.0-7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5, including all values and subranges therebetween. The buffer may contain other components such as a salt (e.g., sodium, potassium, and / or calcium salts). In particular embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, pH 4.5.

[0246] An exemplary, nonlimiting process for making an mRNA-LNP composition involves mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled homogeneous manner, where the ratio of lipids:mRNA is maintained throughout the mixing process. In this illustrative example, the mRNA is presented in an aqueous buffer containing citric acid monohydrate, tri-sodium citrate dihydrate, and sodium chloride. The mRNA solution is added to the solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). The lipid mixture of four lipids (e.g., acationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid) is dissolved in ethanol. The aqueous mRNA solution and the ethanol lipid solution are mixed at a volume ratio of 4: 1 in a “T” mixer with a near “pulseless” pump system. The resultant mixture is then subjected for downstream purification and buffer exchange. The buffer exchange may be achieved using dialysis cassettes or a TFF system. TFF may be used to concentrate and buffer-exchange the resulting nascent LNP immediately after formation via the T-mix process. The diafiltration process is a continuous operation, keeping the volume constant by adding appropriate buffer at the same rate as the permeate flow.Adjuvants

[0247] In some embodiments, the immunogenic composition or vaccine of the present disclosure comprises an adjuvant. In other embodiments, the immunogenic composition or vaccine of the present disclosure does not contain an adjuvant. Similarly, in some embodiments, the immunogenic composition or vaccine of the present disclosure can be administered with an adjuvant to boost the immune response. In other embodiments, the immunogenic composition or vaccines can be administered without an adjuvant. As used herein, the term “adjuvant” refers to a substance or combination of substances that may be used to enhance an immune response to an antigen component of a vaccine or immunogenic composition. Adjuvants can include a suspension of minerals (alum, aluminum salts, including, for example, aluminum hydroxide / oxyhydroxide (A100H), aluminum phosphate (AIPO4), aluminum hydroxyphosphate sulfate (AAHS) and / or potassium aluminum sulfate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in mineral oil (for example, Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund’s complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example, see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biological molecules, such as lipids and costimulatory molecules. Exemplary biological adjuvants include, but are not limited to, AS04 (Didierlaurent et al., J. Immunol., 2009, 183:6186-6197), IL-2, RANTES, GM-CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

[0248] In certain embodiments, the adjuvant is a squalene-based adjuvant comprising an oil-in-water adjuvant emulsion comprising at least: squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant, and a hydrophobic nonionic surfactant. In certainembodiments, the emulsion is thermoreversible, optionally wherein about 90% of the population by volume of the oil drops has a size less than about 200 nm.

[0249] In certain embodiments, the polyoxyethylene alkyl ether is of formula CH3-(CH2)X-(O-CH2-CH2)n-OH, in which n is an integer from 10 to 60, and x is an integer from 11 to 17. In certain embodiments, the polyoxyethylene alkyl ether surfactant is polyoxyethylene(12) cetostearyl ether.

[0250] In certain embodiments, about 90% of the population by volume of the oil drops has a size less than about 160 nm. In certain embodiments, about 90% of the population by volume of the oil drops has a size less than about 150 nm. In certain embodiments, about 50% of the population by volume of the oil drops has a size less than about 100 nm. In certain embodiments, about 50% of the population by volume of the oil drops has a size less than about 90 nm.

[0251] In certain embodiments, the adjuvant further comprises at least one alditol, including, but not limited to, glycerol, erythritol, xylitol, sorbitol and mannitol.

[0252] In some embodiments the hydrophilic / lipophilic balance (HLB) of the hydrophilic nonionic surfactant is greater than or equal to about 10. In certain embodiments, the HLB of the hydrophobic nonionic surfactant is less than about 9. In certain embodiments, the HLB of the hydrophilic nonionic surfactant is greater than or equal to about 10 and the HLB of the hydrophobic nonionic surfactant is less than about 9.

[0253] In certain embodiments, the hydrophobic nonionic surfactant is a sorbitan ester, such as sorbitan monooleate, or a mannide ester surfactant. In certain embodiments, the amount of squalene is between about 5% and about 45%. In certain embodiments, the amount of polyoxyethylene alkyl ether surfactant is between about 0.9% and about 9%. In certain embodiments, the amount of hydrophobic nonionic surfactant is between about 0.7% and about 7%. In certain embodiments, the adjuvant comprises: i) about 32.5% of squalene, ii) about 6.18% of polyoxyethylene(12) cetostearyl ether, iii) about 4.82% of sorbitan monooleate, and iv) about 6% of mannitol.

[0254] In certain embodiments, the adjuvant further comprises an alkylpolyglycoside and / or a cryoprotective agent, such as a sugar, in particular dodecylmaltoside and / or sucrose.

[0255] In certain embodiments, the adjuvant comprises AF03, as described in Klucker et al., J. Pharm. Sci., 2012, 101(12):4490-4500, which is hereby incorporated by reference in its entirety. In certain embodiments, the adjuvant comprises a liposome-based adjuvant, such as SPAM.SPAM is a liposome-based adjuvant (ASOl-like) containing a toll-like receptor 4 (TLR4) agonist (E6020) and saponin (QS21).

[0256] In some embodiments, the vaccine or immunogenic composition does not comprise an adjuvant. In certain embodiments, the one or more mRNA molecules encapsulated in a LNP may serve to adjuvate one or more of the variants of the EBV gB disclosed herein in the vaccine or immunogenic composition. See e.g., Shirai et al., Vaccines, 2020, 8(433): 1-18. In other embodiments, the vaccine or immunogenic composition further comprises an adjuvant.Administration

[0257] The immunogenic compositions or vaccines of the present disclosure can be formulated for administration in any way known in the art of drug delivery, for example, orally, parenterally, intravenously, intramuscularly, subcutaneously, intradermally, transdermally, intrathecally, submucosally, sublingually, rectally, vaginally, etc. In some embodiments, the immunogenic composition or vaccine of the present disclosure is formulated for sublingual administration, intramuscular administration, intradermal administration, subcutaneous administration, intravenous administration, intranasal administration, administration by inhalation, or intraperitoneal administration.

[0258] In some embodiments, the immunogenic composition or vaccine of the present disclosure is formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular. In some embodiments, the immunogenic composition or vaccine of the present disclosure is formulated for sublingual administration. In some embodiments, the immunogenic composition or vaccine is formulated for intramuscular injection. The immunogenic composition or vaccine of the present disclosure may also be formulated for intranasal or inhalation administration. The immunogenic composition or vaccine of the present disclosure can also be formulated for any other intended route of administration.

[0259] In some embodiments, the immunogenic composition or vaccine of the present disclosure is formulated for intradermal injection, intranasal administration or intramuscular injection. General considerations in the formulation and manufacture of pharmaceutical agents for administration by these routes may be found, for example, in Remington’s Pharmaceutical Sciences, 19thed., Mack Publishing Co., Easton, PA, 1995; incorporated herein by reference. At present the oral or nasal spray or aerosol route (e.g., by inhalation) are most commonly used to deliver therapeutic agents directly to the lungs and respiratory system. In some embodiments, theimmunogenic composition or vaccine of the present disclosure is administered using a device that delivers a metered dosage of the vaccine or immunogenic composition. Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patent No. 4,886,499, U.S. Patent No. 5,190,521, U.S. Patent No. 5,328,483, U.S. Patent No. 5,527,288, U.S. Patent No. 4,270,537, U.S. Patent No.5,015,235, U.S. Patent No. 5,141,496, U.S. Patent No. 5,417,662, all of which are incorporated herein by reference. Intradermal compositions may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO 1999 / 34850, incorporated herein by reference, and functional equivalents thereof.

[0260] Also suitable are jet injection devices which deliver liquid vaccines or immunogenic compositions to the dermis via a liquid jet injector or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis. Jet injection devices are described for example in U.S. Patent No. 5,480,381, U.S. Patent No. 5,599,302, U.S. Patent No. 5,334,144, U.S. Patent No. 5,993,412, U.S. Patent No. 5,649,912, U.S. Patent No. 5,569,189, U.S. Patent No.5,704,911, U.S. Patent No. 5,383,851, U.S. Patent No. 5,893,397, U.S. Patent No. 5,466,220, U.S. Patent No. 5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Patent No. 5,520,639, U.S. PatentNo. 4,596,556, U.S. Patent No. 4,790,824, U.S. PatentNo.4,941,880, U.S. Patent No. 4,940,460, WO1997 / 37705, and WO1997 / 13537, all of which are incorporated herein by reference. Additionally, conventional syringes may be used in the classical Mantoux method of intradermal administration.

[0261] Preparations for parenteral administration typically include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0262] The immunogenic compositions or vaccines of the present disclosure may be packaged in a container, such as a prefilled syringe, a vial, or an autoinjector. In some embodiments, theimmunogenic compositions or vaccines of the present disclosure are packaged in a prefilled syringe. In some embodiments, the immunogenic compositions or vaccines of the present disclosure are packaged in a vial. In some embodiments, the immunogenic compositions or vaccines of the present disclosure are packaged in an autoinjector. In other embodiments, the immunogenic compositions or vaccines of the present disclosure are packaged cartridges for patient-friendly autoinjector and infusion pump devices.

[0263] Prefilled syringes provide several advantages over other types of packages, such as convenience, affordability, accuracy, sterility, and safety. Accordingly, in some embodiments, provided herein is a pre-filled syringe comprising about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of any of the immunogenic compositions or vaccines disclosed herein.Methods of Use

[0264] Also provided herein are methods of administering the immunogenic compositions or vaccines described herein to a subject. The methods may be used to vaccinate a subject to prevent an EBV infection in the subject, to decrease the subject’s likelihood of getting an EBV infection, or to reduce the subject’s likelihood of getting serious illness from an EBV infection. Likewise, the present disclosure provides any of the vaccine or immunogenic compositions described herein for use in vaccinating a subject against an EBV infection. Also disclosed is use of any of the immunogenic compositions as described herein for the manufacture of a vaccine for use in vaccinating a subject against an EBV infection. In some embodiments, the vaccination method or use comprises administering to a subject in need thereof an immunologically effective amount of any of the immunogenic compositions or vaccines described herein.

[0265] As used herein, the term “immunologically effective amount” or “therapeutically effective amount” means an amount sufficient to immunize a subject. In some embodiments, the immunologically effective amount or therapeutically effective amount is capable of eliciting protective immunity against an infectious disease, which include, but are not limited to, an increase of antibody titers and / or T cell immunity against an infectious disease. In some embodiments, an immunologically effective amount or therapeutically effective amount of the vaccine or immunogenic composition as disclosed herein increases protective immunity in a subject by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%,including all values and subranges therebetween, when compared with a subject who is not administered with the vaccine or immunogenic composition as disclosed herein.

[0266] Accordingly, in some embodiments, the disclosure provides a method of immunizing a subject comprising administering to the subject in need thereof any of the vaccines or immunogenic compositions described herein. In some embodiments, the disclosure provides a method of immunizing a subject comprising administering to the subject in need thereof an immunologically effective amount any of the vaccines or immunogenic compositions described herein. As used herein, “immunize” or “immunizing” means to induce in a subject a protective immune response against an EBV infection. Likewise, the present disclosure provides any of the vaccine or immunogenic compositions described herein for use in immunizing a subject against an EBV infection. Also disclosed is use of any of the immunogenic compositions as described herein, for the manufacture of a vaccine for use in immunizing a subject against an EBV infection.

[0267] In some embodiments, the method or use prevents EBV infection or disease caused by the EBV infection in the subject. In some embodiments, the method or use decreases the subject’s likelihood of getting an EBV infection by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, including all values and subranges therebetween, when compared with a subject who is not administered with the vaccine or immunogenic composition as disclosed herein. In some embodiments, the method or use reduces the subject’s likelihood of getting serious illness from the EBV infection by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, including all values and subranges therebetween, when compared with a subject who is not administered with the vaccine or immunogenic composition as disclosed herein. In some embodiments, the method or use raises a protective immune response in the subject. In some embodiments, the protective immune response is an antibody response.

[0268] Also provided, in some embodiments, is a method of reducing one or more symptoms of an EBV infection comprising administering to a subject in need thereof any of the vaccines or immunogenic compositions described herein. In some embodiments, provided herein is a methodof reducing one or more symptoms of an EBV infection comprising administering to a subject in need thereof a prophylactically effective amount of any of the vaccines or immunogenic compositions described herein.

[0269] The present disclosure provides any of the vaccine or immunogenic compositions described herein for use in reducing one or more symptoms of an EBV infection. Also disclosed is any of the immunogenic compositions as described herein, for the manufacture of a vaccine for use in reducing one or more symptoms of an EBV infection in a subject.

[0270] In some embodiments, the method or use of the present disclosure reduces one or more symptoms of an EBV infection by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, including all values and subranges therebetween, when compared with a subject who is not administered with the vaccine or immunogenic composition as disclosed herein.

[0271] In some embodiments, the vaccine or immunogenic composition, and an optional adjuvant, may be administered prior to or after development of one or more symptoms of the EBV infection. That is, in some embodiments, the vaccines or immunogenic compositions described herein may be administered prophylactically to prevent the EBV infection or ameliorate the symptoms of a potential EBV infection.

[0272] In some embodiments, the subject is at risk of infection if the subject will be in contact with other individuals or other animals known or suspected to have been infected with an EBV infection and / or if the subject will be present in a location in which EBV infection is known or thought to be prevalent or endemic. In some embodiments, the vaccine or immunogenic composition is administered to a subject suffering from an EBV infection, or the subject is displaying one or more symptoms commonly associated with an EBV infection. In some embodiments, the subject is known or believed to have been exposed to an EBV infection.

[0273] Vaccines or immunogenic compositions in accordance with the present disclosure may be administered in any amount or dose appropriate to achieve a desired outcome. In some embodiments, the desired outcome is induction of a lasting adaptive immune response against the EBV. In some embodiments, the desired outcome is reduction in intensity, severity, and / or frequency, and / or delay of onset of one or more symptoms associated with EBV infection. Insome embodiments, the desired outcome is to provide vaccines or immunogenic compositions with consistent RNA quality. The dose required may vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition being used and its mode of administration.

[0274] In some embodiments, the vaccines or immunogenic compositions described herein are administered to subjects, wherein the subjects can be any member of the animal kingdom. In some embodiments, the subject is a non-human animal. In some embodiments, the non-human subject is a mammal (e.g., a ferret, a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig).

[0275] In some embodiments, the vaccines or immunogenic compositions described herein are administered to a human subject. In some embodiments, a human subject is 6 months of age or older, 6 months through 35 months of age, at least two years of age, at least 3 years of age, 36 months through 8 years of age, 9 years of age or older, at least 6 months of age and less than 5 years of age, at least 6 months of age and less than 18 years of age, or at least 3 years of age and less than 18 years of age. In some embodiments, the human subject is an infant (less than 36 months). In some embodiments, the human subject is a child or adolescent (less than 18 years of age). In some embodiments, the human subject is a child of at least 6 months of age and less than 5 years of age. In some embodiments, the human subject is at least 5 years of age and less than 60 years of age. In some embodiments, the human subject is at least 5 years of age and less than 65 years of age. In some embodiments, the human subject is elderly (at least 60 years of age or at least 65 years of age). In some embodiments, the human subject is a non-elderly adult (at least 18 years of age and less than 65 years of age or at least 18 years of age and less than 60 years of age).

[0276] The methods and uses of the vaccines or immunogenic compositions described herein include administration of a single dose to a subject (i.e., no booster dose). In some embodiments, the methods and uses of the vaccines or immunogenic compositions described herein include prime-boost vaccination strategies. Prime-boost vaccination comprises administering a priming vaccine or immunogenic composition and then, after a period of time has passed, administering to the subject a boosting vaccine or immunogenic composition. The immune response is “primed” upon administration of the priming vaccine or immunogenic composition and is “boosted” upon administration of the boosting vaccine or immunogenic composition. The priming vaccine or immunogenic composition can include a vaccine or immunogenic composition as described hereinand an optional adjuvant. Likewise, the boosting vaccine or immunogenic composition can include a vaccine or immunogenic composition as described herein and an optional adjuvant. The priming vaccine or immunogenic composition can be, but need not be, the same as the boosting vaccine or immunogenic composition. Administration of the boosting vaccine or immunogenic composition is generally weeks or months after administration of the priming vaccine or immunogenic composition, preferably about 2-3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks. In certain embodiments, the recipient of the primeboost vaccination is a naive subject, typically a naive infant or child.

[0277] The vaccine or immunogenic composition can be administered using any suitable route of administration, including, for example, parenteral delivery, as discussed above. In some embodiments, the vaccine or immunogenic composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.Other Applications

[0278] The variants of the EBV gB disclosed herein may have other applications, such as use in an in vitro method for preparation of a trimeric EBV gB complex. Accordingly, provided herein is an in vitro method of preparing a trimeric EBV gB complex, the method comprising expressing an artificial nucleic acid molecule encoding any of the variants of the EBV gB disclosed herein in a host cell to prepare the trimeric EBV gB complex. In some embodiments, the artificial nucleic acid molecule encoding the variant of the EBV gB is part of a vector. In other embodiments, the expression of the variant of the EBV gB is by culturing the host cell in cell culture medium. Thus, in some embodiments, provided herein is an in vitro method of preparing a trimeric EBV gB complex, the method comprising culturing the host cell in a cell culture medium, and expressing the trimeric EBV gB complex. In some embodiments, the in vitro method disclosed herein further comprises a step of purifying the trimeric EBV gB complex from the cell culture medium.

[0279] In certain embodiments, the trimeric EBV gB complex prepared according to the in vitro method disclosed herein is more stable in prefusion conformation as compared to a trimeric EBV gB complex prepared from a control EBV gB, such as a wild-type EBV gB, without the one or more cysteine substitutions disclosed herein. In some embodiments, stability in prefusion conformation is measured by negative stain electron microscopy.

[0280] In certain embodiments, the trimeric EBV gB complex prepared according to the in vitro method disclosed herein is more immunogenic as compared to a trimeric EBV gB complexprepared from a control EBV gB, such as a wild-type EBV gB, without the one or more cysteine substitutions disclosed herein. Immunogenicity can be measured using any methods known in the art.First Set of Representative Embodiments of the Present Disclosure

[0281] Embodiment 1. A variant of an Epstein-Barr virus (EB V) glycoprotein B (gB), wherein the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, and wherein the at least two cysteine substitutions are located in two regions of the EBV gB selected from a first region located at amino acid residues 49-56 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, a second region located at amino acid residues 525-529 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, and a third region located at amino acid residues 631-637 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0282] Embodiment 2. The variant of embodiment 1, wherein the modified EBV gB ectodomain shares at least 95% sequence identity to the ectodomain of the EBV gB.

[0283] Embodiment 3. The variant of embodiment 1 or 2, wherein the cysteine substitution located in the first region of the EBV gB comprises S55C, L53C, or S54C, the cysteine substitution located in the second region of the EBV gB comprises Q527C, V525C, S526C, or V529C, and the cysteine substitution located in the third region of the EBV gB comprises L632C, N635C, I633C, E634C, or D637C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0284] Embodiment 4. The variant of any one of embodiments 1-3, wherein the at least two cysteine substitutions comprise Q527C and L632C, S55C and N635C, L53C and D637C, S54C and S526C, V525C and L632C, S526C and N635C, or Q527C and E634C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0285] Embodiment 5. The variant of any one of embodiments 1-4, wherein the variant further comprises at least two additional cysteine substitutions relative to the EBV gB, and wherein one of the at least two additional cysteine substitutions is located in a fourth region located at amino acid residues 170-177 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 and another one of the at least two additional cysteine substitutions is located in a fifth region located at amino acid residues 560-567 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0286] Embodiment 6. The variant of embodiment 5, wherein the additional cysteine substitution located in the fourth region of the EBV gB comprises G172C, L174C, A175C, or G177C, and wherein the additional cysteine substitution located in the fifth region of the EBV gB comprises D564C or N563C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0287] Embodiment 7. The variant of embodiment 5 or 6, wherein the at least two additional cysteine substitutions comprise G172C andD564C, orL174C andN563C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0288] Embodiment 7a. The variant of any one of embodiments 1-7, wherein the variant comprises at least six cysteine substitutions, and wherein the at least six cysteine substitutions comprise S55C, G172C, Q527C, D564C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0289] Embodiment 8. The variant of any one of embodiments 1-7 and 7a, wherein the variant further comprises at least two, at least four, or at least six further cysteine substitutions relative to the EBV gB.

[0290] Embodiment 9. A variant of an Epstein-Barr virus (EBV) glycoprotein B (gB), wherein the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, wherein the at least two cysteine substitutions are selected from Q527C, L632C, S55C, N635C, G172C, D564C, L53C, S54C, R61C, S63C, I90C, L174C, A175C, G177C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, V529C, N563C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, I633C, E634C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, and R675C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, wherein the at least two cysteine substitutions form a disulfide bridge in the modified EBV gB ectodomai...

Claims

CLAIMS1. A variant of an Epstein-Barr virus (EBV) glycoprotein B (gB), wherein the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, and wherein the at least two cysteine substitutions are located in two regions of the EBV gB selected from a first region located at amino acid residues 49-56 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, a second region located at amino acid residues 525-529 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, and a third region located at amino acid residues 631-637 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

2. The variant of claim 1, wherein the modified EBV gB ectodomain shares at least 95% sequence identity to the ectodomain of the EBV gB.

3. The variant of claim 1 or 2, wherein the cysteine substitution located in the first region of the EBV gB comprises S55C, L53C, or S54C, the cysteine substitution located in the second region of the EBV gB comprises Q527C, V525C, S526C, or V529C, and the cysteine substitution located in the third region of the EBV gB comprises L632C, N635C, I633C, E634C, or D637C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

4. The variant of any one of claims 1-3, wherein the at least two cysteine substitutions comprise Q527C and L632C, S55C and N635C, L53C and D637C, S54C and S526C, V525C and L632C, S526C and N635C, or Q527C and E634C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

5. The variant of any one of claims 1-4, wherein the variant further comprises at least two additional cysteine substitutions relative to the EBV gB, and wherein one of the at least two additional cysteine substitutions is located in a fourth region located at amino acid residues 170-177 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 and another one of the at least two additional cysteine substitutions is located in a fifth region located at amino acid residues 560-567 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

6. The variant of claim 5, wherein the additional cysteine substitution located in the fourth region of the EBV gB comprises G172C, L174C, A175C, or G177C, and wherein the additional cysteine substitution located in the fifth region of the EBV gB comprises D564C or N563C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

7. The variant of claim 5 or 6, wherein the at least two additional cysteine substitutions comprise G172C and D564C, or L174C and N563C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

8. The variant of any one of claims 1-7, wherein the variant comprises at least six cysteine substitutions, and wherein the at least six cysteine substitutions comprise S55C, G172C, Q527C, D564C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

9. The variant of any one of claims 1-8, wherein the variant further comprises at least two, at least four, or at least six further cysteine substitutions relative to the EBV gB.

10. A variant of an Epstein-Barr virus (EBV) glycoprotein B (gB), wherein the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, wherein the at least two cysteine substitutions are selected from Q527C, L632C, S55C, N635C, G172C, D564C, L53C, S54C, R61C, S63C, I90C, L174C, A175C, G177C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, V529C, N563C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, I633C, E634C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, and R675C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, wherein the at least two cysteine substitutions form a disulfide bridge in the modified EBV gB ectodomain, and wherein, if the variant comprises cysteine substitutions G172C and D564C, A175C and V529C, or G322C and D478C, the variant further comprises at least two additional cysteine substitutions relative to the EBV gB.

11. The variant of claim 10, wherein the modified EBV gB ectodomain shares at least 95% sequence identity to the ectodomain of the EBV gB.

12. The variant of claim 11, wherein the at least two cysteine substitutions comprise Q527C and L632C, S55C and N635C, G172C and D564C, L53C and D637C, S54C and S526C, R61C and T621C, S63C and T621C, I90C and T630C, L174C and N563C, A175C and V529C, A175C and E634C, G177C and E634C, K214C and I633C, T225C and N635C, G227C and T591C, T229C and N606C, V318C and G477C, D320C and D478C, G322C and D478C, G322C and A480C, G322C and A482C, T323C and A482C, G376C and T624C, S389C and F463C, I500C and I660C, N501C and F661C, K517C and L657C, G520C and Y594C, V525C and L632C, S526C and N635C, Q527C and E634C, L580C and Y644C, T581C and Y644C, T585C and Q669C, E586C and A668C, G659C and Y664C, or R675C and A651C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

13. The variant of claim 12, wherein the at least two cysteine substitutions comprise Q527C and L632C, S55C and N635C, G172C and D564C, L53C and D637C, S54C and S526C, K517C and L657C, Q527C and E634C, or T581 and Y644C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

14. The variant of any one of claims 10-13, wherein the variant comprises at least four cysteine substitutions selected from Q527C, L632C, S55C, N635C, G172C, D564C, L53C, S54C, R61C, S63C, I90C, L174C, A175C, G177C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, V529C, N563C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, I633C, E634C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, and R675C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, wherein the at least four cysteine substitutions form two disulfide bridges in the modified EBV gB ectodomain.

15. The variant of claim 14, wherein the at least four cysteine substitutions comprise:a) S55C, Q527C, L632C, and N635C;b) G172C, Q527C, D564C, and L632C;c) S55C, G172C, D564C, and N635C;d) L53C, S55C, N635C, and D637C;e) L53C, Q527C, L632C, and D637C;f) S55C, G177C, E634C, and N635C;g) S55C, K517C, N635C, and L657C;h) S55C, Q527C, E634C, and N635C;i) S55C, L580C, N635C, and Y644C;j) S55C, T581C, N635C, and Y644C;k) G172C, A175C, V529C, and D564C;l) G172C, G322C, A480C, and D564C;m) L174C, V318C, G477C, andN563Cn) A175C, G322C, A480C, and V529C;o) G177C, Q527C, L632C, and E634C;p) K517C, Q527C, L632C, and L657C;q) Q527C, L580C, L632C, and Y644C; orr) Q527C, T581C, L632C, and Y644C,as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

16. The variant of claim 15, wherein the at least four cysteine substitutions comprise S55C, Q527C, L632C, and N635C, or G172C, Q527C, D564C, and L632C, or S55C, G172C, D564C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

17. The variant of any one of claims 10-16, wherein the variant comprises at least six cysteine substitutions selected from Q527C, L632C, S55C, N635C, G172C, D564C, L53C, S54C, R61C, S63C, I90C, L174C, A175C, G177C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, V529C, N563C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, I633C, E634C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, and R675C, as indexed by reference to the amino acid sequence of SEQ ID NO:1, wherein the at least six cysteine substitutions form three disulfide bridges in the modified EBV gB ectodomain.

18. The variant of claim 17, wherein the at least six cysteine substitutions comprise:a) S55C, G172C, Q527C, D564C, L632C, and N635C;b) S54C, S55C, S526C, Q527C, L632C, and N635C;c) S55C, L174C, Q527C, N563C, L632C, and N635C;d) S55C, G177C, Q527C, L632C, E634C, and N635C;e) S55C, V318C, G477C, Q527C, L632C, andN635C;f) S55C, D320C, D478C, Q527C, L632C, and N635C; org) G172C, A175C, G322C, A480C, V529C, and D564C,as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

19. The variant of claim 18, wherein the at least six cysteine substitutions comprise:a) S55C, G172C, Q527C, D564C, L632C, and N635C; orb) S54C, S55C, S526C, Q527C, L632C, and N635C,as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

20. The variant of any one of claims 10-19, wherein the variant comprises at least eight cysteine substitutions selected from Q527C, L632C, S55C, N635C, G172C, D564C, L53C, S54C, R61C, S63C, I90C, L174C, A175C, G177C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, V529C, N563C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, I633C, E634C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, and R675C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, wherein the at least eight cysteine substitutions form four disulfide bridges in the modified EBV gB ectodomain.

21. The variant of claim 20, wherein the at least eight cysteine substitutions comprise:a) S55C, G172C, G322C, D478C, Q527C, D564C, L632C, and N635C;b) S55C, G172C, G322C, A480C, Q527C, D564C, L632C, N635C; orc) S55C, L174C, V318C, G477C, Q527C, N563C, L632C, andN635C as indexed by reference to the amino acid sequence of SEQ ID NO:

122. The variant of any one of claims 1-21, further comprising one or more substitutions at amino acid positions W112, Y113, W193, L194, 1195, or W196, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

23. The variant of claim 22, wherein the one or more substitutions comprise W112H, Y113R, W193R, L194V, I195E, and / or W196A.

24. The variant of claim 23, comprising the substitutions W112H, Y113R, W193R, L194V, I195E, and W 196 A.

25. The variant of any one of claims 1-24, wherein amino acid residues 428-434 of the variant, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, comprise the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

26. The variant of any one of claims 1-25, comprising:i) a signal peptide on the N-terminus;ii) a trimerization domain (foldon), a StrepII-tag, a His-tag, a C-tag, and / or a protease cleavage site on the C-terminus; andiii) one or more linkers.

27. The variant of claim 26, wherein:i) the signal peptide comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7;ii) the trimerization domain comprises the amino acid sequence of SEQ ID NO: 8; iii) the StrepII-tag comprises the amino acid sequence of SEQ ID NO: 9;iv) the His-tag comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: ii;v) the C-tag comprises the amino acid sequence of SEQ ID NO: 13; and / orvi) the protease cleavage site is a human rhinovirus (HRV) 3C protease cleavage site comprising the amino acid sequence of SEQ ID NO: 14, a Factor X protease cleavage site comprising the amino acid sequence of SEQ ID NO: 15, or a thrombin cleavage site comprising the amino acid sequence of SEQ ID NO: 16.

28. The variant of any one of claims 1-27, comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

29. A trimeric EBV gB complex, comprising three copies of the variant of any one of claims 1-28.

30. An artificial nucleic acid encoding the variant of any one of claims 1-28.

31. The artificial nucleic acid of claim 30, wherein the artificial nucleic acid is a mRNA.

32. The artificial nucleic acid of claim 30 or 31, wherein the artificial nucleic acid comprises at least one chemically modified nucleotide and / or a phosphorothioate bond.

33. A vector comprising the artificial nucleic acid of any one of claims 30-32.

34. The vector of claim 33, wherein the vector is a messenger RNA (mRNA) production vector.

35. A host cell comprising the vector of claim 33 or 34.

36. An Epstein-Barr virus-like particle (VLP) comprising the variant of any one of claims 1-28.

37. The Epstein-Barr VLP of claim 36, further comprising one or more additional EBV glycoproteins selected from gH / gL, gp42, gp350, and / or gp220, and / or T cell antigens BZLF1,BMRF1, EBNA3a, EBNA3b, EBNA3c, LMP2, BRFL1, BMLF1, and / or EBNA-1, or a combination thereof.

38. A composition comprising the variant of any one of claims 1-28, the trimeric EBV gB complex of claim 29, the artificial nucleic acid of any one of claims 30-32, the vector of claim 33 or 34, or the Epstein-Barr VLP of claim 36 or 37.

39. The composition of claim 38, wherein the composition is an immunogenic composition.

40. An artificial messenger ribonucleic acid (mRNA) encoding a variant of an Epstein-Barr virus (EBV) glycoprotein B (gB), wherein the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, and wherein the at least two cysteine substitutions are located in two regions of the EBV gB selected from a first region located at amino acid residues 49-56 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, a second region located at amino acid residues 525-529 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, and a third region located at amino acid residues 631-637 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

41. The artificial mRNA of claim 40, wherein the modified EBV gB ectodomain shares at least 95% sequence identity to the ectodomain of the EBV gB.

42. The artificial mRNA of claim 40 or 41, wherein the cysteine substitution located in the first region of the EBV gB comprises S55C, L53C, or S54C, the cysteine substitution located in the second region of the EBV gB comprises Q527C, V525C, S526C, or V529C, and the cysteine substitution located in the third region of the EBV gB comprises L632C, N635C, I633C, E634C, or D637C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

43. The artificial mRNA of any one of claims 40-42, wherein the at least two cysteine substitutions comprise Q527C andL632C, S55C andN635C, L53C andD637C, S54C and S526C, V525C and L632C, S526C and N635C, or Q527C and E634C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

44. The artificial mRNA of any one of claims 40-43, wherein the variant further comprises at least two additional cysteine substitutions relative to the EBV gB, and wherein one of the at least two additional cysteine substitutions is located in a fourth region located at amino acid residues 170-177 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 and another one of the at least two additional cysteine substitutions is located in a fifth region located at amino acid residues 560-567 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

45. The artificial mRNA of claim 44, wherein the additional cysteine substitution located in the fourth region of the EBV gB comprises G172C, L174C, A175C, or G177C, and wherein the additional cysteine substitution located in the fifth region of the EBV gB comprises D564C or N563C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

46. The artificial mRNA of claim 44 or 45, wherein the at least two additional cysteine substitutions comprise G172C and D564C, or L174C and N563C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

47. The artificial mRNA of any one of claims 40-46, wherein the variant comprises at least six cysteine substitutions, and wherein the at least six cysteine substitutions comprise S55C, G172C, Q527C, D564C, L632C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

48. The artificial mRNA of any one of claims 40-47, wherein the variant further comprises at least two, at least four, or at least six further cysteine substitutions relative to the EBV gB.

49. An artificial messenger ribonucleic acid (mRNA) encoding a variant of an Epstein-Barr virus (EBV) glycoprotein B (gB), wherein the variant comprises a modified EBV gB ectodomain comprising at least two cysteine substitutions relative to the EBV gB, wherein the at least two cysteine substitutions are selected from Q527C, L632C, S55C, N635C, G172C, D564C, L53C, S54C, R61C, S63C, I90C, L174C, A175C, G177C, Y198C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C,N501C, K517C, G520C, V525C, S526C, V529C, N563C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, I633C, E634C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, R675C, S712C, I771C, and H802C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, wherein the at least two cysteine substitutions form a disulfide bridge in the modified EBV gB ectodomain, and wherein, if the variant comprises cysteine substitutions G172C and D564C, A175C and V529C, or G322C and D478C, the variant further comprises at least two additional cysteine substitutions relative to the EBV gB.

50. The artificial mRNA of claim 49, wherein the modified EBV gB ectodomain shares at least 95% sequence identity to the ectodomain of the EBV gB.

51. The artificial mRNA of claim 50, wherein the at least two cysteine substitutions comprise Q527C and L632C, S55C and N635C, G172C and D564C, L53C and D637C, S54C and S526C, R61C and T621C, S63C and T621C, I90C and T630C, L174C and N563C, A175C and V529C, A175C and E634C, G177C and E634C, Y198C and S712C, K214C and I633C, T225C and N635C, G227C and T591C, T229C andN606C, V318C and G477C, D320C and D478C, G322C and D478C, G322C and A480C, G322C and A482C, T323C and A482C, G376C and T624C, S389C and F463C, I500C and I660C, N501C and F661C, K517C and L657C, G520C and Y594C, V525C and L632C, S526C and N635C, Q527C and E634C, L580C and Y644C, T581C and Y644C, T585C and Q669C, E586C and A668C, G659C and Y664C, R675C and A651C, or I771C and H802C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

52. The artificial mRNA of claim 51, wherein the at least two cysteine substitutions comprise Q527C and L632C, S55C and N635C, G172C and D564C, L53C and D637C, S54C and S526C, K517C and L657C, Q527C and E634C, or T581 and Y644C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

53. The artificial mRNA of any one of claims 40-52, wherein the variant comprises at least four cysteine substitutions selected from Q527C, L632C, S55C, N635C, G172C, D564C, L53C, S54C, R61C, S63C, I90C, L174C, A175C, G177C, Y198C, K214C, T225C, G227C, T229C,V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, V529C, N563C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, I633C, E634C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, R675C, S712C, I771C, and H802C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, wherein the at least four cysteine substitutions form two disulfide bridges in the modified EBV gB ectodomain.

54. The artificial mRNA of claim 53, wherein the at least four cysteine substitutions comprise:a) S55C, Q527C, L632C, and N635C;b) G172C, Q527C, D564C, and L632C;c) S55C, G172C, D564C, and N635C;d) L53C, S55C, N635C, and D637C;e) L53C, Q527C, L632C, and D637C;f) S55C, G177C, E634C, and N635C;g) S55C, K517C, N635C, and L657C;h) S55C, Q527C, E634C, and N635C;i) S55C, L580C, N635C, and Y644C;j) S55C, T581C, N635C, and Y644C;k) G172C, A175C, V529C, and D564C;l) G172C, G322C, A480C, and D564C;m) L174C, V318C, G477C, andN563Cn) A175C, G322C, A480C, and V529C;o) G177C, Q527C, L632C, and E634C;p) K517C, Q527C, L632C, and L657C;q) Q527C, L580C, L632C, and Y644C; orr) Q527C, T581C, L632C, and Y644C,as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

55. The artificial mRNA of claim 54, wherein the at least four cysteine substitutions comprise S55C, Q527C, L632C, and N635C, G172C, Q527C, D564C, and L632C, or S55C, G172C, D564C, and N635C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

56. The artificial mRNA of any one of claims 40-55, wherein the variant comprises at least six cysteine substitutions selected from Q527C, L632C, S55C, N635C, G172C, D564C, L53C, S54C, R61C, S63C, I90C, L174C, A175C, G177C, Y198C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, V529C, N563C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, I633C, E634C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, R675C, S712C, I771C, and H802C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, wherein the at least six cysteine substitutions form three disulfide bridges in the modified EBV gB ectodomain.

57. The artificial mRNA of claim 56, wherein the at least six cysteine substitutions comprise:a) S55C, G172C, Q527C, D564C, L632C, and N635C;b) S54C, S55C, S526C, Q527C, L632C, and N635C;c) S55C, L174C, Q527C, N563C, L632C, and N635C;d) S55C, G177C, Q527C, L632C, E634C, and N635C;e) S55C, V318C, G477C, Q527C, L632C, andN635C;f) S55C, D320C, D478C, Q527C, L632C, and N635C; org) G172C, A175C, G322C, A480C, V529C, and D564C,as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

58. The artificial mRNA of claim 57, wherein the at least six cysteine substitutions comprise:a) S55C, G172C, Q527C, D564C, L632C, and N635C; orb) S54C, S55C, S526C, Q527C, L632C, and N635C,as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

59. The artificial mRNA of claim 45 or 58, wherein the variant further comprises at least two cysteine substitutions comprise Y198C and S712C and / or I771C and H802C.

60. The artificial mRNA of any one of claims 40-59, wherein the variant comprises at least eight cysteine substitutions selected from Q527C, L632C, S55C, N635C, G172C, D564C, L53C,S54C, R61C, S63C, I90C, L174C, A175C, G177C, Y198C, K214C, T225C, G227C, T229C, V318C, D320C, G322C, T323C, G376C, S389C, F463C, G477C, D478C, A480C, A482C, I500C, N501C, K517C, G520C, V525C, S526C, V529C, N563C, L580C, T581C, T585C, E586C, T591C, Y594C, N606C, T621C, T624C, T630C, I633C, E634C, D637C, Y644C, A651C, L657C, G659C, I660C, F661C, Y664C, A668C, Q669C, R675C, S712C, I771C, and H802C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, wherein the at least eight cysteine substitutions form four disulfide bridges in the modified EBV gB ectodomain.

61. The artificial mRNA of claim 60, wherein the at least eight cysteine substitutions comprise:a) S55C, G172C, G322C, D478C, Q527C, D564C, L632C, and N635C;b) S55C, G172C, G322C, A480C, Q527C, D564C, L632C, N635C; orc) S55C, L174C, V318C, G477C, Q527C, N563C, L632C, andN635C,as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

62. The artificial mRNA of claim 61, wherein the variant further comprises at least two cysteine substitutions comprise Y198C and S712C and / or I771C and H802C.

63. The artificial mRNA of any one of claims 40-62, wherein the variant further comprises substitutions R837T and R838T, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

64. The artificial mRNA of any one of claims 40-63, comprising a nucleic acid sequence having at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57.

65. The artificial mRNA of claim 64, comprising the nucleic acid sequence of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57.

66. The artificial mRNA of any one of claims 40-65, comprising a 5'-cap structure and / or a 3'-poly(A) sequence.

67. The artificial mRNA of any one of claims 40-66, comprising at least one chemically modified nucleotide and / or a phosphorothioate bond.

68. The artificial mRNA of claim 67, wherein the at least one chemically modified nucleotide comprises a pseudouridine, a 2'-fluoro ribonucleotide, or a 2'-methoxy ribonucleotide, optionally wherein the pseudouridine is a N1 -methylpseudouridine.

69. A composition comprising the artificial mRNA of any one of claims 40-68 encapsulated in a lipid nanoparticle (LNP).

70. The composition of claim 69, wherein the LNP comprises a cationic lipid.

71. The composition of claim 70, wherein the cationic lipid comprises or is OF-02, cKK-ElO, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, (4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), or IM-001.

72. The composition of claim 70 or 71, wherein the LNP further comprises a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol -based lipid, and a helper lipid.

73. The composition of claim 72, wherein:a) the PEGylated lipid comprises or is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000); and / orb) the cholesterol-based lipid comprises or is cholesterol; and / orc) the helper lipid comprises or is dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

74. The composition of claim 72 or 73, wherein:a) the cationic lipid is present at a molar ratio between about 35% and about 55%; b) the PEGylated lipid is present at a molar ratio between about 0.25% and about 2.75%;c) the cholesterol-based lipid is present at a molar ratio between about 20% and about 45%; andd) the helper lipid is present at a molar ratio between about 5% and about 35%, wherein all of the molar ratios are relative to the total lipid content of the LNP.

75. The composition of claim 74, wherein:a) the cationic lipid is present at a molar ratio of about 40%;b) the PEGylated lipid is present at a molar ratio of about 1.5%;c) the cholesterol-based lipid is present at a molar ratio of about 28.5%; and d) the helper lipid is present at a molar ratio of about 30%,wherein all of the molar ratios are relative to the total lipid content of the LNP.

76. The composition of any one of claims 69-75, wherein the composition is an immunogenic composition.

77. A vaccine comprising the composition of claim 39 or 76, and a pharmaceutically acceptable carrier.

78. The vaccine of claim 77, further comprising an adjuvant.

79. A method of immunizing a subject, the method comprising administering to the subject in need thereof the vaccine of claim 77 or 78.

80. The method of claim 79, wherein the method prevents an Epstein-Barr virus infection in the subject, decreases the subject’s likelihood of getting an Epstein-Barr virus infection, or reduces the subject’s likelihood of getting serious illness from an Epstein-Barr virus infection.

81. The method of claim 79 or 80, wherein the subject is a human.

82. The method of any one of claims 79-81, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

83. A method of reducing one or more symptoms of an Epstein-Barr virus infection, the method comprising administering to a subject in need thereof the vaccine of claim 77 or 78.

84. An in vitro method of preparing a trimeric EBV gB complex, the method comprising culturing the host cell of claim 35 in a cell culture medium, and expressing the EBV gB complex.

85. The in vitro method of claim 84, further comprising a step of purifying the trimeric EBV gB complex from the cell culture medium.

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