Compositions and methods for inducing an immune response against epstein-barr virus

Abstract

The present disclosure relates to nucleic acid molecules (e.g., self-replicating RNAs (srRNAs) expressing one or more antigenic determinants derived from Epstein-Barr virus (EBV)), recombinant cells containing the nucleic acid molecules and pharmaceutical compositions, and uses of such srRNA molecules, recombinant cells and compositions for eliciting pharmacodynamic effects in a subject. Methods for preventing and / or treating a variety of health conditions associated with EBV infection are also provided.

Description

Cross-references to related applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 615,206, filed December 27, 2023. The disclosure of the above-cited application is hereby expressly incorporated herein by reference in its entirety (including any accompanying drawings). Merging of sequence lists

[0002] The material in the attached sequence list is incorporated herein by reference. The attached sequence list XML file, named 058462_519001WO_SequenceListing.XML, was created on December 19, 2024, and is approximately 266,240 bytes in size. Technical Field

[0003] This disclosure relates to the fields of molecular virology and immunology, and particularly to nucleic acid molecules (such as self-replicating RNA (srRNA)) expressing antigenic determinants derived from Epstein-Barr virus (EBV), pharmaceutical compositions containing said nucleic acid molecules, and recombinant cells, as well as the use of such srRNA molecules, recombinant cells, and compositions to induce pharmacodynamic effects in subjects. Methods for the prevention and / or treatment of various health conditions associated with EBV infection are also provided. Background Technology

[0004] Epstein-Barr virus (EBV) infects most people worldwide and persists for life due to the complex interplay between multiple types of lytic and latent infections. While often asymptomatic, EBV is a causative agent for several types of cancer and is strongly associated with autoimmune and inflammatory diseases such as multiple sclerosis. Specifically, it is one of the most common human viruses, infecting approximately 95% of the adult population globally and is known to be associated with two types of B-cell lymphoma (Burkit lymphoma and Hodgkin lymphoma). EBV can also infect epithelial cells and is associated with nasopharyngeal carcinoma. EBV is transmitted through bodily fluids, most commonly saliva, and primarily infects young children and adolescents (seropositivity rates of approximately 50% and 89%, respectively). It is the leading cause of infectious mononucleosis (IM) in the United States, accounting for over 90% of the approximately 1-2 million cases annually. Infectious mononucleosis can debilitate patients for weeks to months and, in some cases, can lead to hospitalization and splenic rupture. EBV infection is associated with the development and progression of certain lymphoproliferative disorders and cancers, as well as an increased risk of autoimmune diseases, including multiple sclerosis, an autoimmune disease of the central nervous system.

[0005] Currently, there are no FDA-approved vaccines against EBV on the market. Therefore, there is an urgent need in the art to develop methods and agents, such as prophylactic and therapeutic vaccines, for EBV and EBV-related diseases. The technical problem upon which this disclosure is based is to identify alternative and / or improved compositions, means, and methods that can be used for therapeutic and prophylactic vaccination against EBV infection and EBV-related diseases (such as infectious mononucleosis, multiple sclerosis, and EBV-positive cancers). Summary of the Invention

[0006] This disclosure generally relates to the development of immunotherapeutic agents, such as recombinant nucleic acid constructs and pharmaceutical compositions comprising said nucleic acid constructs, for use in the prevention and management of various health conditions, such as proliferative disorders, inflammatory diseases, autoimmune diseases, and cancer. In particular, as described in more detail below, some embodiments of this disclosure provide nucleic acid constructs containing sequences encoding a modified genome or self-replicating RNA (srRNA) (e.g., replicons) of an alphavirus genus, wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of said modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct containing one or more antigenic determinants derived from Epstein-Barr virus (EBV) proteins. Recombinant cells and transgenic animals engineered to comprise one or more nucleic acid constructs disclosed herein are also disclosed, as well as pharmaceutical compositions comprising one or more of the following: (a) nucleic acid constructs of this disclosure, and (b) recombinant cells of this disclosure. In some embodiments, this disclosure also provides EBV vaccines capable of eliciting anti-EBV antibody responses and / or potent neutralizing antibody responses and / or potent anti-EBV T-cell responses. Certain aspects of this disclosure further provide compositions and methods for inducing pharmacodynamic effects in subjects with such needs, and / or for preventing and / or treating various health conditions, including inflammatory diseases, autoimmune diseases, proliferative disorders (such as cancer), and viral infections.

[0007] In one aspect of this disclosure, a nucleic acid construct is provided comprising a nucleic acid sequence encoding a modified alphavirus genome or self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of said modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct comprising one or more antigenic determinants derived from Epstein-Barr virus (EBV).

[0008] Non-limiting exemplary embodiments of the nucleic acid constructs disclosed herein may include one or more of the following features. In some embodiments, one or more antigenic determinants are derived from one or more EBV polypeptides / antigens, said EBV polypeptides / antigens being encoded by a cleavage gene, a pre-latent gene, a latent gene, an early gene, an immediate early gene, a late gene, a latent reactivation gene, any variant thereof, or any combination thereof. In some embodiments, the EBV latent gene is LMP2, BKRF1, BYRF1, BLRF3 / BERF1, BERF2a / b, BERF3 / 4, BamHI-W, BNLF1 (LMP1), BARTs, or EBER1 / 2. In some embodiments, the EBV latent gene is LMP2, BNLF1 (LMP1), BKRF1 (EBNA1), BLRF3 / BERF1 (EBNA3A-C, -LP), or BYRF1 (EBNA2). In some embodiments, the EBV immediate early gene is BZLF1, BRLF1, or BMLF1. In some implementations, the early gene is BRRF1, BORF2, BaRF1, BXLF1, BGLF5, BLLF3, BKRF3, BALF5, BMRF1, BALF2, BSLF1, BBLF2 / 3, BBLF4, BSLF2, BHRF1, BALF1, BARF1, BGLF4, BFRF1, BHLF1, BHLF2, or BNLF2a. In some embodiments, the late EBV gene is BLLF1 (gp350 / 220), BNRF1, BPLF1, BOLF1, BVRF1, BBLF1, BGLF1, BSRF1, BRRF2, BDLF2, BKRF4, BcLF1, BDLF1, BFRF3, BLRF2, BdRF1, BBRF1, BVRF2, BGLF2, BORF1, BLRF1 (gN), BLLF1 (gp350 / 220), BZLF2, BKRF2 (gp25, gL), BBRF3 (gM), BXLF2 (gp85, gH), BILF1, BILF2, BALF4 (gp100, gB), BDLF3, BMRF2, BALF3, or BCRF1. In some embodiments, the EBV cleavage gene is BLLF1 (gp350 / 220), BXLF2 (gp85, gH), BALF4 (gp100, gB), BSLF1 (gp42), or BKRF2 (gp25, gL). In some embodiments, the EBV pre-latent gene is BYRF1 (EBNA2).In some embodiments, the one or more EBV genes are selected from the group consisting of BLLF1 (gp350 / 220), BZLF1, BXLF2 (gp85, gH), BKRF2 (gp25, gL), LMP2, and any combination thereof. In some embodiments, the LMP2 gene encodes isotype LMP2A. In some embodiments, the LMP2 gene encodes isotype LMP2B. LMP2B lacks exon 1 in its amino acid sequence but otherwise overlaps with LMP2A.

[0009] In some embodiments of this disclosure, the coding sequence of the polypeptide construct includes the coding sequences of the following genes: (a) BZLF1 or a variant thereof; (b) BLLF1 (gp350 / 220) or a variant thereof; (c) LMP2B or a variant thereof; (d) gH and gL or variants thereof; (e) BZLF1, LMP2B, gH, gL, and BLLF1 (gp350 / 220) or any variant thereof; (f) gH, gL, and BLLF1 (gp350 / 220) or any variant thereof; or (g) BZLF1 and LMP2B or any variant thereof. In some embodiments, BLLF1 (gp350 / 220) or a variant thereof lacks a functional transmembrane (TM) domain. In some embodiments, gH or a variant thereof lacks a functional transmembrane (TM) domain. In some implementations, the BZLF1 or its variants lack (i) a functional nuclear localization sequence (NLS) and / or (ii) a functional inverse activation (TA) domain.

[0010] In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34.

[0011] In some embodiments of this disclosure, the coding sequences of the antigenic determinants are operatively linked to each other within a single open reading frame (i.e., a polycistronic ORF). In some embodiments of this disclosure, the coding sequences of the antigenic determinants are operatively linked to each other within two or more ORFs. In some embodiments, the coding sequences of the antigenic determinants are operatively linked to each other via one or more adapters. In some embodiments, the one or more adapters comprise coding sequences for autoproteolytic peptides or internal ribosome entry sites (IRES). In some embodiments, the autoproteolytic peptide comprises one or more autoproteolytic cleavage sequences derived from calcium-dependent serine endonuclease (furin), porcine cephalovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis virus A (ERAV) 2A (E2A), *Typha laniceps* virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), muscular muscular dermatitis virus 2A (BmIFV2A), or combinations thereof. In some embodiments, the polypeptide construct of this disclosure includes one or more P2A autologous proteolytic cleavage sequences. In some embodiments, the internal ribosome entry site (IRES) is derived from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, virociritis virus IRES, cricket paralysis virus IRES, aphid virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, microRNA virus IRES, encephalomyocorticoid virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES. In some embodiments, the internal ribosome entry site (IRES) is derived from encephalomyocorticoid virus (EMCV).

[0012] In some embodiments, the one or more EBV antigenic determinants are EBV type 1 (EBV-1) or EBV type 2 (EBV-2) or a combination thereof. In some embodiments, the one or more EBV antigenic determinants are from virulent or non-virulent EBV strains. In some embodiments, the one or more EBV antigenic determinants are derived from the AG876 strain, Akata strain, Alaska strain, B95-8 strain, China 2 strain, CVI988 strain, GD1 strain, GD2 strain, GP202 strain, Hina1 strain, HKNPC strain, M81 strain, Mediterranean strain, Mutu strain, P3HR1 strain, Raji strain, SNU-719 strain, YCCEL1 strain, or a combination thereof.

[0013] In some embodiments, the modified alphavirus genome or srRNA does not include a nucleic acid sequence encoding a viral structural protein. In some embodiments, the nucleic acid sequence encoding the polypeptide construct is operatively linked to a promoter sequence. In some embodiments, the promoter sequence is a 26S subgenomic (sg) promoter. In some embodiments, the srRNA is a capped srRNA containing a 5'-cap. In some embodiments, the capped srRNA is a co-transcribed capped srRNA. In some embodiments, the capped srRNA is an enzymatically capped srRNA.

[0014] In some embodiments, the modified alphavirus genome or srRNA is an alphavirus belonging to the VEEV / EEEV group, the SFV group, or the SINV group. In some embodiments, the alphavirus is Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Chikungunya virus (CHIKV), Madariaaga virus (MADV), Western equine encephalitis virus (WEEV), or Sindbis virus (SINV).

[0015] In some embodiments, the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35-56.

[0016] In one aspect, this document provides recombinant cells comprising nucleic acid constructs as described herein. In a related aspect, this document provides cell cultures comprising at least one recombinant cell as described herein and a culture medium. Non-limiting exemplary embodiments of the recombinant cells disclosed herein may include one or more of the following features: In some embodiments, the recombinant cells are eukaryotic cells. In some embodiments, the recombinant cells are animal cells. In some embodiments, the animal cells are vertebrate cells or invertebrate cells. In some embodiments, the recombinant cells are insect cells. In some embodiments, the recombinant insect cells are mosquito cells. In some embodiments, the animal cells are mammalian cells. In some embodiments, the recombinant cells are selected from the group consisting of: monkey kidney CV1 cells transformed with SV40, human embryonic kidney cells (HEK), young hamster kidney cells (BHK) or their derivatives, mouse supporting cells, monkey kidney cells, human cervical cancer cells, canine kidney cells, buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumors, TRI cells, FS4 cells, Chinese hamster ovary cells (CHO), African green monkey kidney cells, human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NSO mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human B cells, human epithelial cells, human T cells, human dendritic cells, human macrophages, human RAW 264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

[0017] On the other hand, this document provides transgenic animals comprising nucleic acid constructs and / or recombinant cells as described herein. In some embodiments, the transgenic animal is a vertebrate or invertebrate. In some embodiments, the animal is an insect. In some embodiments, the animal is a mammal. In some embodiments, the mammal is a non-human mammal.

[0018] In one aspect, this document provides compositions (e.g., pharmaceutical compositions) comprising pharmaceutically acceptable excipients and one or more of the following: (a) nucleic acid constructs as described herein; and (b) recombinant cells as described herein.

[0019] Non-limiting exemplary embodiments of the pharmaceutical compositions disclosed herein may include one or more of the following features. In some embodiments, the composition comprises a nucleic acid construct as described herein and a pharmaceutically acceptable excipient. In some embodiments, the composition comprises recombinant cells as described herein and a pharmaceutically acceptable excipient. In some embodiments, the composition is formulated together with a delivery medium into a delivery system, wherein the delivery system comprises liposomes, viral replicon particles (VRPs), lipid-based nanoparticles (LNPs), polymer nanoparticles, physiological buffers, microspheres, immunostimulatory complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof. In some embodiments, the LNP delivery system comprises cationic lipids, ionizable cationic lipids, anionic lipids, and / or neutral lipids. In some embodiments, the lipids of the LNP delivery system are present in a lipid-to-RNA mass ratio of about 100:1 to about 4:1. In some embodiments, the delivery system comprises lipid-based nanoparticles with an average diameter of less than 1000 nm, less than 500 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 75 nm, less than 50 nm, or less than 25 nm. In some embodiments, the LNP delivery system comprises lipid-based nanoparticles with an average diameter of about 1-1000 nm, about 1-1000 nm, about 1-500 nm, about 1-250 nm, about 25-200 nm, about 25-100 nm, about 35-75 nm, or about 25-60 nm.

[0020] In some embodiments, the pharmaceutical compositions disclosed herein are immunogenic compositions. In some embodiments, the compounds are formulated as vaccines or adjuvants. In some embodiments, the compositions are formulated for one or more of the following routes of administration: intranasal, intrathecal, transdermal, intraperitoneal, intramuscular, intratracheal, intranodular, intratumoral, intra-articular, intravenous, subcutaneous, intravaginal, intraocular, rectal, and oral. In some specific embodiments, the compositions are formulated for intramuscular administration.

[0021] On the other hand, this document provides a method for inducing a pharmacodynamic effect in a subject with such a need, the method comprising administering to the subject a composition comprising: (a) a nucleic acid construct as described herein; (b) recombinant cells as described herein; and / or (c) a pharmaceutical composition as described herein. Non-limiting exemplary examples of the methods disclosed herein for inducing a pharmacodynamic effect may include one or more of the following features. In some embodiments, the pharmacodynamic effect includes one or more of the following: immunogenic effect, biomarker response, therapeutic effect, prophylactic effect, desired effect, undesired effect, adverse effect, and disease model effect. In some embodiments, the pharmacodynamic effect includes prevention of viral infection and / or reduction of viral load in the subject. In some embodiments, the pharmacodynamic effect includes prevention and / or reduction of disease severity in the subject. In some embodiments, the pharmacodynamic effect includes inducing an immune response in the subject. In some embodiments, the immune response includes one or more of the following: anti-EBV antibody response, neutralizing antibody response, and anti-EBV T cell response.

[0022] On the other hand, this document provides methods for preventing and / or treating health conditions in subjects who require such treatment, the methods comprising prophylactically or therapeutically administering to the subject a composition comprising: (a) a nucleic acid construct as described herein; (b) recombinant cells as described herein; and / or (c) a pharmaceutical composition as described herein. Non-limiting exemplary examples of the methods disclosed herein for preventing and / or treating health conditions may include one or more of the following features: In some embodiments, the administered composition elicits an immune response in the subject. In some embodiments, the administered composition results in an increased production of interferon in the subject. In some embodiments, the subject has or is suspected of having an EBV-related disease. In some embodiments, the EBV-related disease is infectious mononucleosis (IM), an autoimmune disease, or cancer.

[0023] In some embodiments, the composition is administered to the subject as a single agent for prevention or therapy (monotherapy), alone or as a first therapy in combination with at least one additional therapy. In some embodiments, the at least one additional therapy is selected from the group consisting of: chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery.

[0024] On the other hand, this document provides kits for inducing pharmacodynamic effects, inducing immune responses, and / or for preventing and / or treating health conditions, said kits comprising: (a) nucleic acid constructs as described herein; (b) recombinant cells as described herein; and / or (c) pharmaceutical compositions as described herein, and instructions for performing one or more methods disclosed herein. In some embodiments, the health condition is an EBV-related disease. In some embodiments, the EBV-related disease is infectious mononucleosis (IM), an autoimmune disease, or cancer.

[0025] Each aspect and implementation described herein can be used together unless explicitly or clearly excluded in the context of that implementation or aspect.

[0026] The foregoing description of the invention is illustrative only and is not intended to be limiting in any way. Other aspects, embodiments, objects, and features of this disclosure, in addition to the illustrative embodiments and features described herein, will become fully apparent from the drawings, detailed description, and claims. Attached Figure Description

[0027] Figure 1 This is an illustration of a non-limiting example of a multi-gene EBV vaccine design according to certain embodiments of this disclosure, wherein the nucleic acid sequences encoding viral structural proteins of the original virus have been completely deleted. The multi-gene EBV vaccine design described in this figure contains native 5' UTRs and 3' UTRs derived from alphavirus, and also contains the heterologous gene of interest (GOI), which is a multi-gene expression cassette under the control of the 26S subgenomic promoter. The coding sequences for the non-structural proteins nsP1, nsP2, nsP3, and nsP4 are indicated.

[0028] Figure 2 Schematic diagrams of twenty-two (22) exemplary nucleic acid constructs expressing EBV antigenic determinants derived from one or more EBV peptides / antigens, according to some embodiments of this disclosure, are shown. The number of EBV antigens and their order of arrangement in the antigen cassette are shown in each construct. While some constructs contain coding sequences of multiple peptides operatively linked to each other within a single open reading frame (e.g., in a polycistronic ORF), other constructs comprise coding sequences of multiple peptides configured as two OFRs, the coding sequences of which are interconnected by sequences at internal ribosome entry sites (IRES). P2A: Autoproteolytic peptide sequence from porcine cyclophosphamide-1 2A. Except for gH in Rep-678, Figure 2 All gp350 and gH coding sequences described in the text lack a functional transmembrane domain (-TM). Figure 2The coding sequence of BZLF1 described in the figure lacks (i) a functional nuclear localization sequence (NLS) and (ii) a functional transactivation (TA) domain. In this figure, the EBV peptide, the autologous proteolytic peptide P2A sequence, and the internal ribosome entry site (IRES) are shown in the N-terminal to C-terminal direction (i.e., 5' to 3' direction) of their respective coding sequences.

[0029] Figure 3 The results of the experiments conducted are summarized schematically, which aimed to illustrate srRNA-based bioactivity, as measured by electroporating BHK-21 cells with replication intermediate dsRNA using various EBV srRNA vaccines. In this figure, the frequency of dsRNA+ cells for each construct is depicted.

[0030] Figure 4 The results of experiments conducted are summarized schematically to illustrate protein expression of three of five antigens from various multi-gene or single-gene srRNA vaccines in electroporated BHK-21 cells. In these experiments, protein expression was detected by flow cytometry using antibodies against gp350 and gH / gL complexes. The mean fluorescence intensity (MFI) of the fluorescently labeled antibodies was used as a measure of protein expression.

[0031] Figure 5 The results of the experiments conducted, designed to illustrate BZLF1 protein expression in various single-gene and multi-gene srRNA vaccines in electroporated BHK-21 cells, are illustrated graphically. In these experiments, protein expression was detected by Western blotting of whole-cell lysates from electroporated BHK-21 cells using a BZLF1 antibody. Actin expression was used as a loading control for the samples.

[0032] Figures 6A-6B The results of the experiments conducted are summarized schematically, and the experiments were designed to demonstrate that srRNA-based vaccines can generate antibody responses against encoded EBV antigens in vivo (ELISA). In these experiments, antibody responses against three EBV surface antigens were measured by total IgG ELISA 13 days after two doses of the srRNA-based vaccine (1 µg each): gH / gL complex ( Figure 6A ) and gp350 ( Figure 6B Using serum from immunized mice, the IgG antibody response to EBV binding to the EBV protein gp350 and gH / gL complex was measured by a direct ELISA method. Standard mouse antibodies for each EBV antigen were used to quantify the response.

[0033] Figures 7A-7DThe results of the experiments conducted are summarized schematically, demonstrating that srRNA-based vaccines can generate T-cell responses to encoded EBV antigens in vivo (ELISpot). In these experiments, T-cell responses to five encoded antigens were measured by IFNγ ELISpot 13 days after two doses of the srRNA-based vaccine (1 µg): gH and gL ( Figure 7A ), gp350 ( Figure 7B LMP2B Figure 7C ) and BZLF1 ( Figure 7D T cell responses were measured by stimulating intact spleen cells with a pool of peptides corresponding to each encoded antigen and by detecting IFNγ secretion using a commercially available ELISpot kit.

[0034] Figure 8 The results of the experiments conducted are summarized schematically to demonstrate that srRNA-based EBV vaccines can generate a neutralizing antibody response against EBV in vivo. Detailed Implementation

[0035] This document provides, among other things, viral expression systems comprising self-replicating RNA (srRNA) based on alphaviruses with superior expression potential. These systems are suitable for expressing heterologous molecules, such as antigens and antigenic determinants from Epstein-Barr virus (EBV), for therapeutic purposes in human health conditions or diseases, including post-transplant lymphoproliferative disorder (PTLD), autoimmune diseases, inflammatory diseases, and cancer. These srRNA-based expression systems address the complexities of the EBV lifecycle and infection. For example, some embodiments of this disclosure relate to nucleic acid constructs (such as expression constructs and vectors) containing a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct containing multiple antigenic determinants derived from EBV. Additionally, recombinant cells genetically engineered to include one or more nucleic acid constructs disclosed herein are also provided. Biomaterials and recombinant products derived from such recombinant cells are also within the scope of this application. Compositions and methods are also provided for (i) modulating pharmacodynamic effects and preventing and / or treating EBV-related health conditions in subjects who require such treatment. definition

[0036] Unless otherwise defined, all technical terms, symbols, and other scientific terms or specialized expressions used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this application pertains. In some cases, terms with their commonly understood meanings are defined herein for clarity and / or for ease of reference, and such definitions contained herein are not necessarily to be construed as representing a material difference from the meaning commonly understood in the art. Many of the techniques and procedures described or mentioned herein are well understood by one of ordinary skill in the art and are typically employed by one of ordinary skill in the manner of practice.

[0037] Unless the context clearly specifies otherwise, the singular forms “a / an / a kind of (a) or (an)” and “the” include plural indicators. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternative forms: “A”, “B”, “A or B”, and “A and B”.

[0038] As used herein, the terms “administration” and “administering” refer to the delivery of a bioactive composition or formulation via a route of administration, including but not limited to intranasal, transdermal, intravenous, intra-arterial, intramuscular, intra-tubercular, intraperitoneal, subcutaneous, intramuscular, oral, vaginal, and local administration, or combinations thereof. The terms include, but are not limited to, administration performed by a medical professional and self-administration.

[0039] The terms “cell,” “cell culture,” and “cell line” refer not only to the specific subject cell, cell culture, or cell line, but also to the offspring or potential offspring of such cells, cell cultures, or cell lines, regardless of the number of transfers or passages in culture. It should be understood that not all offspring are identical to the parent cells. This is because certain modifications can occur in offspring due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), making offspring virtually different from the parent cells, but still included within the scope of the terminology used herein, provided that the offspring retain the same function as the original cell, cell culture, or cell line.

[0040] The term "antigenic determinant" refers to a portion (e.g., fragment or site) of an antigen molecule that is specifically recognized by B or T lymphocytes. An example of an antigenic determinant is an epitope, which interacts with the antigen-binding site of an antigen-binding polypeptide (e.g., a variable region of an antibody molecule) or a T-cell receptor (TCR) when an antigen is presented to the major histocompatibility complex (MHC). B lymphocytes generate a response to foreign antigenic determinants via antibodies, while T lymphocytes are mediators of cell-mediated immunity. Therefore, an antigenic determinant or epitope is that portion of an antigen recognized by an antibody, or, in the case of the MHC, by a T-cell receptor. Epitopes can be linear or conformational, the latter consisting of a continuous amino acid sequence or not. In some embodiments, an epitope may include a determinant that is a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphate group, or a sulfonyl group, and in some embodiments, an epitope may have a specific three-dimensional structural characterization and / or a specific charge characterization. Those skilled in the art will understand that a single antigen may have more than one antigenic determinant or epitope. Therefore, different antibodies can bind to different regions of an antigen and can have different biological effects. The term epitope also refers to the site where B and / or T cells respond to an antigen. It also refers to the portion of the antigen that is bound by an antibody or TCR (such as a region, fragment, or site).

[0041] The terms "effective amount," "therapeutic effective amount," or "pharmaceutical effective amount" in this disclosure generally refer to an amount sufficient, relative to the absence of the composition, to enable the composition to achieve its stated purpose (e.g., to achieve the effect of its administration, to stimulate an immune response, to prevent or treat a disease, or to alleviate one or more symptoms of a disease, disorder, infection, or health condition). An example of an "effective amount" is an amount sufficient to induce treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutic effective amount." "Alleviation" of symptoms means a reduction in the severity or frequency of one or more symptoms, or the elimination of one or more symptoms. The exact amount of the composition (including the “therapeutic effective amount”) will depend on the purpose of treatment and will be determined by someone skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th edition, 2003, edited by Gennaro, Lippincott, Williams & Wilkins).

[0042] The term "construction" refers to a recombinant molecule (e.g., a recombinant nucleic acid or polypeptide) that comprises one or more isolated nucleic acid sequences or amino acid sequences from a heterologous source. For example, a polypeptide construct may be a chimeric polypeptide molecule in which two or more amino acid sequences from different sources are operatively linked together in a single polypeptide construct. Similarly, a nucleic acid construct may be a chimeric nucleic acid molecule in which two or more nucleic acid sequences from different sources are assembled into a single nucleic acid molecule. Thus, representative nucleic acid constructs include any construct containing: (1) a nucleic acid sequence, including regulatory and coding sequences that are not found adjacent to each other in nature (e.g., at least one nucleotide sequence is heterologous relative to at least one other nucleotide sequence thereof), or (2) a sequence encoding a non-naturally adjacent functional RNA molecule or protein moiety, or (3) a portion of a non-naturally adjacent promoter. Representative nucleic acid constructs may include any recombinant nucleic acid molecule, linear or circular, single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source (e.g., plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages), capable of genome integration or autonomous replication, and containing nucleic acid molecules in which one or more nucleic acid sequences have been operatively linked. In some embodiments, the nucleic acid constructs of this disclosure may include essential elements to direct the expression of a nucleic acid sequence of interest also included in the construct. Such elements may include control elements, such as promoters operatively linked to the nucleic acid sequence of interest (e.g., to direct transcription), and optionally include polyadenylated sequences.

[0043] In some embodiments of this disclosure, the nucleic acid construct may be incorporated into a vector. The term "vector" is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. Therefore, the term "vector" encompasses both DNA-based vectors and RNA-based vectors. The term "vector" includes cloning vectors and expression vectors, as well as viral vectors and integration vectors. An "expression vector" is a vector that includes a regulatory region, thereby enabling the expression of DNA sequences and fragments in vivo, in vitro, and / or in vitro. In some embodiments, the vector may include sequences that guide autonomous replication in cells, such as, for example, plasmids (DNA-based vectors) or self-replicating RNA vectors. In some embodiments, the vector may include sequences sufficient to allow integration into the host cell's DNA. Useful vectors include, for example, plasmids (such as DNA plasmids or RNA plasmids), transposons, granules, bacterial artificial chromosomes, and viral vectors. In some embodiments, the vectors of this disclosure may be single-stranded vectors (e.g., ssDNA or ssRNA). In some embodiments, the vectors of this disclosure may be double-stranded vectors (e.g., dsDNA or dsRNA). In some embodiments, the vector is a gene delivery vector. In some implementations, vectors are used as gene delivery media to transfer genes into cells.

[0044] In addition to the components of the construct, the vector may also include, for example, one or more optional markers, one or more replication origins (such as prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the cellular genome. Two or more constructs may be integrated into a single nucleic acid molecule (such as a single vector) or may be integrated into two or more separate nucleic acid molecules (such as two or more separate vectors). An “expression construct” typically includes at least one control sequence operatively linked to a nucleotide sequence of interest. For example, in this manner, a promoter operatively linked to the nucleotide sequence to be expressed is provided in the expression construct for expression in cells. Compositions and methods for preparing and using constructs and cells are known to those skilled in the art for the practice of this disclosure.

[0045] As used herein, the term “operably linked” refers to a physical or functional connection between two or more elements (e.g., a polypeptide sequence or a polynucleotide sequence) that allows them to operate in their intended manner. For example, when used in the context of a nucleic acid molecule or coding and promoter sequences within a nucleic acid molecule described herein, the term “operably linked” means that the coding and promoter sequences are within a frame and at appropriate spatial and distance distances to allow for influence on transcription via the corresponding binding of transcription factors or RNA polymerases. It should be understood that operably linked elements can be continuous or discontinuous (e.g., linked to each other by a linker). Thus, operably linked segments, portions, regions, and domains of polypeptide or nucleic acid molecules disclosed herein can be continuous or discontinuous (e.g., linked to each other by a linker). In the context of a polypeptide construct, “operably linked” refers to a physical connection (e.g., a direct or indirect connection) between amino acid sequences (e.g., different segments, portions, regions, or domains) to provide the described activity of the construct.

[0046] As used herein, the term "portion" refers to a fraction. With respect to a particular structure (such as a polynucleotide sequence, amino acid sequence, or protein), the term "portion" can refer to a continuous or discontinuous portion of said structure. For example, a portion of an amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the amino acids in said amino acid sequence. Alternatively or additionally, if said portion is discontinuous, then said discontinuous portion consists of 2, 3, 4, 5, 6, 7, 8, or more portions of a structure (e.g., a domain of a protein), each portion being a continuous element of the structure. For example, the discontinuous portion of the amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8 or more (e.g., no more than 4) portions of the amino acid sequence, wherein each portion contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or at least 30 consecutive amino acids of the amino acid sequence.

[0047] When the term "recombinant" is used to refer to cells, nucleic acids, proteins, or vectors, it indicates that the cells, nucleic acids, proteins, or vectors have been altered or generated through human intervention, such as through modification by laboratory methods or as a result of laboratory methods. Thus, for example, recombinant proteins and nucleic acids include proteins and nucleic acids generated by laboratory methods. Recombinant proteins may include amino acid residues not found in the natural (non-recombinant or wild-type) form of the protein, or may include amino acid residues that have been modified (e.g., labeled). The term may include any modification to the sequence of a peptide, protein, or nucleic acid. Such modifications may include: any chemical modification of the sequence of a peptide, protein, or nucleic acid, including chemical modification of one or more amino acids, deoxynucleotides, or ribonucleotides; the addition, deletion, and / or substitution of one or more amino acids in a peptide or protein; the generation of fusion proteins, such as fusion proteins containing antibody fragments; and the addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence. When the term "recombinant" is used to refer to cells, it is not intended to include naturally occurring cells, but covers cells that have been engineered / modified to contain or express peptides or nucleic acids that would not be present in the cell if not engineered / modified.

[0048] In the context of two or more nucleic acids or proteins, the term "percentage of sequence identity," as used herein, refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acids (e.g., approximately 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity when compared and aligned over a comparison window or specified region to obtain the maximum correspondence), as measured by manual alignment and visual inspection using the BLAST or BLAST 2.0 sequence alignment algorithm with the default parameters described below. See, for example, the NCBI website ncbi.nlm.nih.gov / BLAST. Such sequences are subsequently referred to as "substantially identical." This definition also relates to or can be applied to complements of query sequences. This definition includes sequence alignment performed using the BLAST algorithm, where the parameters of the algorithm are selected to give the maximum match across the full length of the respective reference sequence. This definition also includes sequences with deletions and / or additions, as well as those with substitutions. Sequence identity can be calculated within a region of at least about 20 amino acids or nucleotides, or within a region of 10–100 amino acids or nucleotides, or over the full length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and FASTA (Atschul et al., J Mol Biol 215:403, 1990). Sequence identity can also be measured using sequence analysis software with its default parameters, such as the sequence analysis software package from the Genetics Computer Group at the University of Wisconsin-Madison Biotechnology Center (1710 University Avenue, Madison, Wisconsin, 53705). Other methods that may be suitable for determining amino acid sequence similarity or identity include those that rely on position-specific structure scoring matrices (P3SMs) that incorporate structure prediction scores from Rosetta, and those that are based on length-normalized edit distance, such as those previously described, for example, in Setcliff et al., Cell Host & Microbe 23(6), May 2018.

[0049] As used herein, the term "pharmaceuticalally acceptable excipient" means any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration to a subject of a compound of interest. Therefore, "pharmaceuticalally acceptable excipient" can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term "pharmaceuticalally acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Supplemental active compounds (e.g., antibiotics and other therapeutic agents) may also be incorporated into the composition.

[0050] As used herein, "subject" or "individual" includes animals, such as humans (e.g., individual humans) and non-human animals. In some embodiments, "subject" or "individual" is a patient under the care of a physician. Thus, the subject can be a human patient or individual who has, is at risk of having, or is suspected of having one or more symptoms of a health condition of interest (e.g., EBV-related cancer or infection) and / or a health condition. The subject can also be an individual who is diagnosed at or after diagnosis as being at risk of having a health condition of interest. The term "non-human animal" includes all vertebrates, such as mammals (e.g., rodents, such as mice, non-human primates, and other mammals, such as sheep, dogs, cattle, chickens) and non-mammals (e.g., amphibians, reptiles, etc.).

[0051] It should be understood that the aspects and embodiments of this disclosure described herein include aspects and embodiments that are “comprising,” “composed of,” and “substantially composed of.” As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended, not excluding additional, undescribed elements or method steps. As used herein, “composed of” excludes any element, step, or component not specified in the claimed composition or method. As used herein, “substantially composed of” does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claimed composition or method. The term “comprising,” particularly in the description of the components of a composition or the steps of a method, is understood herein to cover compositions and methods substantially composed of and formed therefrom the said components or steps.

[0052] When numerical ranges are provided, those skilled in the art will understand that all ranges disclosed herein include any and all possible subranges and combinations thereof. Any listed range can be readily considered to adequately describe the same range and can be decomposed into at least equal halves, ternaries, quarters, quintiles, decimals, etc. As a non-limiting example, each range discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. In some embodiments, when numerical ranges are provided, it should be understood that every intermediate value (in tenths of the lower limit) between the upper and lower limits of the range, as well as any other specified value or intermediate value within the range, is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also included in this disclosure, subject to any exact exclusion limits within the stated range. When a described range includes one or two limits, the range excluding any one or both of those included limits is also included in this disclosure. Those skilled in the art will also understand that all terms such as “at most,” “at least,” “greater than,” and “less than” include the numbers stated and refer to the ranges discussed above, which can subsequently be broken down into subranges. Furthermore, as those skilled in the art will understand, the range includes each individual member. Thus, for example, a group having 1-3 items means a group having 1, 2, or 3 items. Similarly, a group having 1-5 items means a group having 1, 2, 3, 4, or 5 items, and so on.

[0053] The term “about” precedes certain ranges of numerical values ​​presented herein, and has the general meaning of approximation. The term “about” is used herein to provide textual support for an exact number that follows, as well as numbers that are close to or approximate to the number that follows the term. In determining whether a number is close to or approximate to a specifically listed number, the unlisted close or approximate number may, in its context, be a number that is substantially equivalent to the specifically listed number. If the degree of approximation is unclear in the context, “about” means within 10% of the provided value, or rounded to the nearest significant figure, in all cases including the provided value. In some embodiments, the term “about” means a specified value ± at most 10%, at most ± 5%, or at most ± 1%.

[0054] Titles, such as (a), (b), (i), etc., exist solely for the purpose of facilitating the reading of the specification and claims. The use of titles in the specification or claims does not require that the steps or elements be performed in alphabetical, numerical, or other order of appearance.

[0055] It should be understood that, for clarity, certain features of this disclosure are described in the context of individual embodiments, or may be combined in a single embodiment. Conversely, for brevity, the various features of this disclosure described in a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments relating to this disclosure are specifically included in this disclosure and disclosed herein as if each combination were separately and explicitly disclosed. Furthermore, this disclosure specifically includes all sub-combinations of various embodiments and their elements, and discloses them herein as if each such sub-combination were separately and explicitly disclosed herein. Epstein-Barr virus

[0056] Epstein-Barr virus (EBV), also known as human herpesvirus 4 (HHV-4), is a common human herpesvirus belonging to the γ family, along with another human virus, Kaposi's sarcoma-associated herpesvirus (KHSV). Unlike alpha and beta herpesviruses, these viruses have the ability to induce cancer. EBV belongs to the γ1 or lymphocryptovirus genus and was identified as the first human oncology virus. EBV can be divided into two main types: EBV type 1 (EBV-1; also known as type A EBV) and EBV type 2 (EBV-2 or type B EBV). These two subtypes have different EBNA-3 genes. Therefore, the two subtypes differ in their conversion and reactivation capabilities. Type 1 is dominant in most parts of the world, but both types are equally prevalent in Africa.

[0057] EBV is associated with a variety of diseases and malignancies. For example, EBV is a cause of infectious mononucleosis (glandular fever) and is associated with certain forms of proliferative disorders such as post-transplant lymphoproliferative disorder (PTLD), autoimmune diseases, inflammatory diseases and cancers such as Hodgkin's lymphoma, Burkitt's lymphoma, nasopharyngeal carcinoma, and health conditions associated with human immunodeficiency virus (HIV) such as hairy leukoplakia and central nervous system lymphoma.

[0058] In particular, EBV infection is associated with a higher risk of developing certain autoimmune diseases, especially dermatomyositis, systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, and multiple sclerosis. EBV infects more than 90% of the global population, but it exists latently in most infected individuals and evades immune surveillance. Approximately 20,000 cancer cases are attributed to EBV each year, such as B-cell malignancies, nasopharyngeal carcinoma (NPC), gastric cancer (GC), and some rare T / NK-cell lymphomas, leukemia, and leiomyosarcoma. Epithelial carcinomas NPC and GC are the leading causes of death among EBV-related malignancies, with EBV+GC being the largest category of EBV-positive cancers, accounting for over 80,000 cases annually.

[0059] Infectious mononucleosis (glandular fever) is an extremely common, self-limiting acute illness associated with primary EBV infection. It is characterized by lymphadenopathy that usually resolves promptly, transient fever, and hepatosplenomegaly. Chronic active EBV infection (CAEBV), although rare, is a serious and fatal condition characterized by an abnormally high EBV DNA load (10³–10⁷ copies / mL). It is currently considered one of the EBV+ T or NK cell lymphoproliferative disorders and can lead to two fatal conditions: hemophagocytic lymphohistiocytosis (HLH) and chemotherapy-resistant lymphoma. EBV life cycle and infection

[0060] Epstein-Barr virus (EBV) exhibits a biphasic life cycle, including a latency and lysis (replication) phase. The typical route of transmission is through bodily fluids (such as saliva), where the oral cavity transmits the virus and infects resting B cells and epithelial cells in the mouth. Primary infection is usually asymptomatic, but 35–50% of adolescents develop infectious mononucleosis (IM) approximately one month after infection, and the virus persists throughout an individual's life. Following acute infection, the virus enters a dormant state due to a strong virus-specific T-cell response. However, when the balance between the virus and the host's immune system is disrupted, EBV can drive malignant transformations of lymphoid and epithelial origin, causing approximately 200,000 deaths annually.

[0061] As a herpesvirus, EBV can cause latent or lytic infection. In epithelial cells, EBV typically replicates through lysis. In B cells, EBV is usually latent for life, with rare and occasional reactivation. During the latent phase, only a few important viral genes are expressed, and viral particle production ceases. The transition from latent to mature stages is governed by a variety of factors. While products encoded by EBV in both stages can play a role in transformation and tumorigenesis, the oncogenic role of latent genes is more extensively documented compared to lytic genes. However, targeting latent EBV using current immunotherapy strategies is challenging, particularly due to reduced antigen expression. Therefore, patients with EBV+ or EBV- tumors often undergo similar treatment regimens.

[0062] Following infection, the virus typically remains latent within host cells. During this latency period, only a few latent genes essential for maintaining and preserving the viral genome are expressed. EBV encodes eight latent genes, and their expression in host cells and / or malignancies defines the EBV latency program. Based on the expression of these eight latent viral genes, viral infection is classified into three main latency programs: Latency III, Latency II, and Latency I / O. Immature B cells infected with EBV exhibit the Latency III program, which allows infected cells to proliferate and expand. The Latency III genes include six EBV nuclear antigens (EBNA1, 2, 3A, 3B, 3C, LP), two latent membrane proteins (LMP1 and LMP2), an EBV-encoded small RNA (EBER), and an EBV-encoded microRNA (miRNA). The LMP2 gene encodes two protein isotypes, LMP2A and LMP2B. LMP2B lacks exon 1 but otherwise overlaps with LMP2A. LMP2B lacks exon 1 in its amino acid sequence, but otherwise overlaps with LMP2A.

[0063] Cells in this latency program are highly immunogenic and can be rapidly cleared by the host immune response, particularly by EBV-specific T cells. In latency II, the expression of EBV genes (i.e., EBNA1, LMP1, and LMP2A / B) is more restricted, resulting in lower immunogenicity. Ultimately, EBV sequentially shuts down the expression of all latent genes in latency I except for EBNA1 and a few EBV-encoded RNAs. Based on the expression of LMP and EBNA2-3, latency II can also be divided into IIa and IIb (IIb is EBNA2-3+LMP-; IIa is EBNA2-3-LMP+). In most individuals, EBV exists quiescently in a subset of memory B cells (<0.005% of B cells in peripheral blood), expressing no viral genes in the latency 0 state, also known as "true latency." Latent EBV genes have been reported to promote tumorigenesis, inhibit apoptosis, and suppress the recognition of infected cells by host immune cells. EBV-related malignancies are associated with different EBV latency programs. Lymphoproliferative disorders typically associated with immunosuppression, such as post-transplant lymphoproliferative disease (PTLD) and lymphomas associated with acquired immunodeficiency syndrome (AID), exhibit latency stage III. Hodgkin's lymphoma, T / NK cell lymphoma, and nasopharyngeal carcinoma (NPC) exhibit latency stage II. Gastric cancer and Burkitt's lymphoma exhibit latency stage I (27, 31). To date, EBV with latency stage 0 has not been associated with any malignancy, possibly due to its dormant state during this period.

[0064] The lysis phase is essential for EBV progeny production and lateral viral spread from host to host, thus representing an indispensable aspect of viral pathogenicity (32). The transition from latency to the lysis cycle can be spontaneous or chemically induced. Some commonly used agents for inducing the lysis cycle include phorbol ester (PMA), sodium butyrate, calcium ionophores, DNA methyltransferase inhibitors (DNMTi), transforming growth factor-β (TGF-β), doxorubicin, and gemcitabine (because these agents are stress-induced chemotherapeutic agents), as well as anti-IgG or anti-IgM as B-cell receptor stimulants. During lysis reactivation, a full-text library of >80 viral genes is temporarily regulated and expressed in three phases: immediate early (IE), early (E), and late (L). The first phase is primarily initiated by BZLF1 (ZEBRA) and BRLF1 (two key EBV immediate early (IE) lysis transcription factors). The function of these two genes promotes their own expression and that of each other, as well as the expression of viral E genes encoding proteins required for viral replication, such as viral DNA polymerase. BZLF1 forms a homodimer via its basic leucine zipper motif and binds to the BZLF1 response element (ZRE) on DNA. Binding of BZLF1 to CpG-methylated DNA leads to the activation of several lethal viral genes silenced by CpG methylation in latent cells. Additionally, binding of BZLF1 to the lysis replication origin (oriLyt) ZRE promotes lysis viral DNA synthesis. Similarly, BRLF1 binds to the BRLF1 response element (RRE) on DNA and has been reported to induce lysis replication via the PI3K and ERK signaling pathways. Both BZLF1 and BRLF1 are crucial for EBV lysis replication because knocking out these genes prevents the transition from latent to lysis phase. Furthermore, overexpression of BZLF1 and BRLF1 in latently infected cells can induce EBV lysis reactivation. This lysis induction leads to a cascade expression of viral genes that promote viral DNA replication and viral production. Following viral replication, late viral genes encoding structural proteins (such as gp350 / 220 encoded by the BLLF1 gene) are expressed. Interestingly, during γ-herpesvirus lytic DNA replication, transcription of late-stage lytic viral genes requires continuous DNA synthesis, while early-stage lytic genes do not. Virosomes can spread viral particles within and between host cells. EBV replicates in latency phases I, II, and III via the proliferation of activated B cells. It has been reported that lytic replication can only be effectively induced starting from latency phase I / O and following extensive methylation of the viral genome. This is because BZLF1 prefers to bind to methylated CpG sequences to initiate the production of infectious particles.

[0065] Currently, there are no FDA-approved vaccines available for the prevention of primary EBV infection and / or EBV-related diseases. Therefore, there is an urgent need in the field to develop methods and agents, such as prophylactic and therapeutic vaccines, for EBV and EBV-related diseases (e.g., infectious mononucleosis and EBV-positive cancers). Alphavirus self-replicating RNA A virus

[0066] In some embodiments of this disclosure, the replicon (e.g., srRNA) is derived from a virus belonging to the genus *Anavirus*. The genus *Anavirus* has been extensively studied, and the life cycle, replication patterns, etc., of these viruses have been well characterized. Anaviruses are small, enveloped RNA viruses with a single-stranded, positive-sense RNA genome. More information on this can be found in Arrigo NC et al., ibid., 2010; Corrin T. et al., Vector-Borne and Zoonotic Diseases, Vol. 21, No. 5, 2021. Furthermore, anaviruses have shown highly efficient replication in animal cells, making them valuable as vectors for the production of proteins and nucleic acids in such cells. Interspecies and inter-individual transmission is primarily via mosquitoes, classifying anaviruses as a type of arbovirus (also known as "arthropod-borne viruses").

[0067] The genus *Anavirus* includes, in particular, Sindbis virus (SINV), Semliki forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV). These are closely related and can infect a wide variety of vertebrates (such as mammals, rodents, fish, birds, and large mammals such as humans and horses) and invertebrates (such as insects). Each of these alphaviruses has a positively polar, single-stranded RNA genome enclosed in a nucleocapsid containing the viral spike protein. Alphavirus particles are enveloped, spherical (though slightly pleomorphic), and have an isometric nucleocapsid. The alphavirus genome is a positively polar, single-stranded RNA, approximately 11-12 kb in length, with 5' and 3' untranslated regions (UTRs) flanking it. The 5' end is capped and the 3' end is polyadenylated. It contains two open reading frames: the first frame (approximately 7 kb) encodes a non-structural protein with enzymatic function, and the second frame (approximately 4 kb) encodes viral structural proteins (such as the capsid protein CP, E1 glycoprotein, E2 glycoprotein, E3 protein, and 6K protein). The non-structural polyprotein (nsP) is cleaved into four distinct proteins (nsP1, nsP2, nsP3, and nsP4), which are essential for the transcription and translation of viral mRNA within the host cell cytoplasm.

[0068] The 5' end two-thirds of the alphavirus genome encodes a variety of non-structural proteins (nsPs) essential for viral RNA transcription and replication. These proteins are translated directly from RNA and, together with cellular proteins, form RNA-dependent RNA polymerases, which are crucial for viral genome replication and subgenomic RNA transcription. Four non-structural proteins (nsP1, nsP2, nsP3, and nsP4) are produced as a single polyprotein, constituting the viral replication mechanism. Polyprotein processing occurs in a highly regulated manner, with cleavage at the P2 / P3 junction affecting the use of the RNA template during genome replication. This site is located at the bottom of a narrow cleavage and is not easily accessible. Once cleaved, nsP3 forms a ring structure around nsP2. These two proteins have a wide contact surface. Mutations in nsP2 that produce non-cytopathic viruses or temperature-sensitive phenotypes accumulate in the P2 / P3 contact region. P3 mutations, located opposite to the non-cytopathic mutations in nsP2, prevent efficient cleavage of P2 / P3. This, in turn, affects RNA infectivity, altering the level of viral RNA production.

[0069] The 3' end third of the genome contains subgenomic RNA, which serves as a template for translating all the structural proteins required to form the viral particle: the core nucleocapsid protein C and the envelope proteins P62 and E1, which are heterodimerized. Viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells via membrane fusion. The subgenomic RNA is transcribed from the p26S subgenomic promoter located at the 3' end of the RNA sequence encoding the nsp4 protein. P62 undergoes proteolytic maturation to E2 and E3, resulting in changes to the viral surface. E1, E2, and sometimes E3, along with the glycoprotein "spikes," together form the E1 / E2 dimer or E1 / E2 / E3 trimer, where E2 extends from the center to the apex, E1 fills the space between the apexes, and E3 (if present) is located at the distal end of the spikes. When the virus is exposed to the acidic environment of the endosome, E1 dissociates from E2 to form the E1 homotrimer, which is essential for driving the fusion step where the cell membrane and viral membrane are joined together. Alphavirus glycoprotein E1 is a type II viral fusion protein, structurally different from type I fusion proteins found in influenza viruses and HIV. The E2 glycoprotein functions through the interaction of its cytoplasmic domain with the nucleocapsid, while its extracellular domain is responsible for binding to cellular receptors. Most alphaviruses lose the peripheral protein E3, but in Semlikie Forest virus, it remains associated with the viral surface.

[0070] According to reports, alphavirus replication occurs on the membrane surface within the host cell. In the first step of the infection cycle, the 5' end of the genomic RNA is translated into polyproteins (nsP1-4) with RNA polymerase activity, which produce a negative strand complementary to the genomic RNA. In the second step, the negative strand is used as a template for the production of two types of RNA: (1) a positive genomic RNA corresponding to the genome of secondary viruses that produce other nsPs through translation and serving as the viral genome; and (2) a subgenomic RNA encoding the structural proteins of the virus that form the infection particle. The ratio of positive genomic RNA to subgenomic RNA is regulated by the self-cleavage of the polyproteins into nsP1, nsP2, nsP3, and nsP4. In practice, viral gene expression occurs in two phases. In the first phase, both the positive and negative genomic strands are synthesized. During the second phase, the synthesis of subgenomic RNA is virtually exclusive, thus leading to the production of a large number of structural proteins. Self-replicating RNA

[0071] As will be understood by those skilled in the art, the term “self-replicating RNA” (srRNA) refers to an RNA molecule containing all the genetic information required to direct its own amplification or self-replication within a permissible cell. Therefore, srRNA is sometimes also referred to as “self-amplifying RNA” (saRNA). In some embodiments, srRNA is a “replicon,” which can be a linear or circular portion of DNA or RNA that replicates sequentially as a unit. Non-limiting examples of replicons include “replicon RNA” or “RNA replicon.” To direct its own replication, srRNA typically (1) encodes a polymerase, replicase, or other protein that may interact with viral or host cell-derived proteins, nucleic acids, or ribonucleoproteins to catalyze the RNA amplification process; and (2) contains cis-acting RNA sequences required for the replication and transcription of subgenomic RNA. These sequences may bind during replication to proteins they encode or to non-encoded cell-derived proteins, nucleic acids, or ribonucleoproteins, or complexes between any of these components.

[0072] In some embodiments of this disclosure, the alphavirus srRNA construct (such as srRNA, saRNA, or RNA replicon molecule) typically contains the following elements: a 5' viral or defective interfering RNA sequence required for cis-replication, a sequence encoding a biologically active alphavirus nonstructural protein (e.g., nsP1, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) for subgenomic RNA (sgRNA), a 3' viral sequence required for cis-replication, and optionally a poly(A) segment. In some cases, a subgenomic promoter (sg) that directs the expression of a heterologous sequence may be included in the srRNA construct of this disclosure.

[0073] Furthermore, the term srRNA (such as srRNA, saRNA, or RNA replicon molecule) generally refers to a molecule with positive polarity or "information" significance, and the length of srRNA may differ from the length of srRNA in any known, naturally occurring alphavirus. In some embodiments of this disclosure, the srRNA does not contain at least a portion of the coding sequence for one or more alphavirus structural proteins; and / or the sequence encoding the structural gene may be substituted with a heterologous sequence. In these cases, when the srRNA is packaged into recombinant alphavirus particles, it may contain one or more sequences, the so-called packaging signals, which are used to initiate interactions with alphavirus structural proteins, thereby leading to particle formation.

[0074] The srRNA constructs disclosed herein typically have a length of at least about 2 kb. For example, the srRNA may have a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb. In some embodiments, the srRNA may have a concentration of about 4 kb to about 20 kb, about 4 kb to about 18 kb, about 5 kb to about 16 kb, about 6 kb to about 14 kb, about 7 kb to about 12 kb, about 8 kb to about 16 kb, about 9 kb to about 14 kb, about 10 kb to about 18 kb, about 11 kb to about 16 kb, about 5 kb to about 18 kb, about 6 kb to about 20 kb, about 5 kb to about 10 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb. The srRNA can have a length of approximately 6 kb to approximately 14 kb. In some embodiments, the srRNA can have a length of approximately 6 kb to approximately 16 kb. The compositions disclosed herein

[0075] As described in more detail below, one aspect of this disclosure relates to nucleic acid constructs containing nucleic acid sequences encoding modified viral genomes or srRNAs, wherein the modified genome or srRNA lacks (e.g., does not include) at least a portion of a nucleic acid sequence encoding one or more structural proteins of the corresponding unmodified viral genome or srRNA. Some embodiments of this disclosure provide a modified alphavirus genome or srRNA containing coding sequences for non-structural proteins nsP1, nsP2, nsP3, and nsP4, but lacking at least a portion or the complete sequence encoding one or more structural proteins. Recombinant cells, cell cultures, and transgenic animals engineered to include nucleic acid constructs as disclosed herein are also provided. This disclosure also provides compositions (e.g., pharmaceutical compositions) comprising one or more of the following: (a) nucleic acid constructs as described herein; and (b) recombinant cells as described herein. A. Nucleic acid constructs

[0076] As described in more detail below, one aspect of this disclosure relates to novel nucleic acid constructs encoding alphavirus genomes or srRNAs as described herein. In some embodiments, the nucleic acid construct comprises a nucleic acid sequence encoding a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of the modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct comprising one or more antigenic determinants derived from Epstein-Barr virus (EBV). For example, in some embodiments, the modified alphavirus genome or srRNA of this disclosure may include one or more deletions, substitutions, and / or insertions of one or more genomic regions in a parental alphavirus genome. In some embodiments, the sequence encoding the srRNA of this disclosure may be operatively linked, for example, under the control of elements desired for expression (such as promoter sequences), thereby allowing expression of the srRNA in host cells, in a subject, or in an ex vivo cell-free expression system.

[0077] Non-limiting exemplary embodiments of the nucleic acid constructs (such as genomes and srRNA constructs) disclosed herein may include one or more of the following features. In some embodiments, the one or more antigenic determinants are derived from one or more EBV peptides / antigens encoded by cleavage genes, pre-latent genes, latent genes, early genes, immediate early genes, late genes, latent reactivation genes, any variants thereof, or any combination thereof. For example, in some embodiments, the peptide construct includes antigenic determinants derived from one or more EBV latent genes. Examples of EBV latent genes suitable for the compositions and methods of this disclosure include BKRF1, BYRF1, BLRF3 / BERF1, BERF2a / b, BERF3 / 4, BamHI-W, BNLF1 (LMP1), LMP2, BART, and EBER1 / 2. In some embodiments, the peptide construct includes an antigenic determinant derived from the EBV latent / pre-latent gene LMP2, a transmembrane protein commonly expressed in latently infected cells. LMP2 has two isotypes (LMP2A and LMP2B). LMP2A is associated with maintaining EBV latency. For example, LMP2A can exclude the B cell receptor (BCR) from lipid rafts to prevent lysis induction. The N-terminal region of LMP2A contains an immune receptor tyrosine-based activation motif (ITAM), which can lead to aberrant cellular signaling in infected cells. In some embodiments, the polypeptide construct includes an antigenic determinant derived from an LMP2B isotype, which typically lacks 119 N-terminal amino acids compared to LMP2A including the ITAM. Without being bound by any particular theory or mechanism of action, in some embodiments of this disclosure, LMP2B is chosen as the vaccine antigen because it is considered to have a lower safety risk compared to LMP2A, while including antigenic determinants that overlap with the remaining homologous sequences between LMP2A and LMP2B.

[0078] In some embodiments, the polypeptide construct comprises antigenic determinants derived from one or more EBV immediate early genes. Non-limiting examples of immediate early genes include BZLF1, BRLF1, and BMLF1. In some embodiments, the polypeptide construct comprises one or more antigenic determinants derived from the EBV immediate early gene BZLF1. In some embodiments, the polypeptide construct comprises antigenic determinants derived from one or more EBV early genes. Suitable EBV early genes include, but are not limited to, BRRF1, BORF2, BaRF1, BXLF1, BGLF5, BLLF3, BKRF3, BALF5, BMRF1, BALF2, BSLF1, BBLF2 / 3, and BBLF4. Further examples of EBV early genes suitable for the compositions and methods disclosed herein include BSLF2, BHRF1, BALF1, BARF1, BGLF4, BFRF1, BHLF1, BHLF2, and BNLF2a.

[0079] In some embodiments, the polypeptide construct includes antigenic determinants derived from one or more late EBV genes. Non-limiting examples of late EBV genes suitable for the compositions and methods of this disclosure include BNRF1, BPLF1, BOLF1, BVRF1, BBLF1, BGLF1, BSRF1, BRRF2, BDLF2, BKRF4, BcLF1, BDLF1, BFRF3, BLRF2, BdRF1, BBRF1, BVRF2, BGLF2, BORF1, and BLRF1 (gN). Further examples of late EBV genes suitable for the compositions and methods disclosed herein include BLLF1 (gp350 / 220), BZLF2, BKRF2 (gp25, gL), BBRF3 (gM), BXLF2 (gp85, gH), BILF1, BILF2, BALF4 (gp100, gB), BDLF3, BMRF2, BALF3, and BCRF1.

[0080] In some embodiments, the polypeptide construct includes antigenic determinants derived from one or more EBV cleavage genes. Suitable early EBV genes include, but are not limited to, BLLF1 (gp350 / 220), BXLF2 (gp85, gH), BALF4 (gp100, gB), BSLF1 (gp42), and BKRF2 (gp25, gL). Those skilled in the art will understand that BALF4 (gp100, gB), BLLF1 (gp350 / 220), and BKRF2 (gp25, gL) can also be classified as late genes. BSLF1 can also be classified as an early gene.

[0081] In some embodiments, the polypeptide construct includes antigenic determinants derived from one or more pre- or late-stage EBV genes. Examples of pre- or late-stage EBV genes suitable for the compositions and methods of this disclosure include, but are not limited to, BNLF1 (LMP1), LMP2, BKRF1 (EBNA1), BLRF3 / BERF1 (EBNA3A-C, -LP), and BYRF1 (EBNA2).

[0082] In some implementations, the polypeptide construct contains one or more antigenic determinants derived from the EBV gene BLLF1, a glycoprotein crucial for the effective infection of resting B cells by EBV. The EBV open reading frame BLLF1 encodes the major envelope glycoproteins gp350 and gp220. The full-length open reading frame of this transcript encodes gp350, while a single splice of the primary transcript deletes 197 codons and in-frames 401 and 699 of gp350 to generate gp220. Gp350 is the most abundant viral protein in the viral envelope. This large protein is extensively glycosylated and localized to multiple subcellular compartments of replicating cells (cytoplasm, endoplasmic reticulum, Golgi apparatus, and plasma membrane). EBV binds to primary B cells via gp350 interactions with CD21 and complement receptor 2 (CR2). Several gp350 domains have been reported to be involved in the formation of a stable complex with CD21, one of which has been identified as a receptor binding site. This polysaccharide-free domain is also recognized by the neutralized gp350-specific antibody 72A. In some embodiments of the compositions and methods disclosed herein, the peptide construct includes one or more antigenic determinants derived from gp220. In some embodiments of the compositions and methods disclosed herein, the peptide construct contains one or more antigenic determinants derived from gp350. In some embodiments of the compositions and methods disclosed herein, the peptide construct contains one or more antigenic determinants derived solely from gp350 and not gp220.

[0083] In some embodiments, the polypeptide construct contains an antigenic determinant derived from the EBV gH-gL complex, which comprises three glycoproteins: gp85, a gH homolog and a product of BXLF2; gp25, a gL homolog and a product of BKRF2; and gp42, a product of BZLF2. The complex behaves similarly in many respects to its counterparts in other herpesviruses. The gH glycoprotein depends on gL for actual processing and transport, and overall, the complex is considered crucial for the virus's ability to fuse with the cell membrane and penetrate the cytoplasm.

[0084] In some embodiments, the polypeptide construct includes antigenic determinants derived from one or more of the following EBV cleavage genes: BLLF1 (gp350 / 220), BXLF2 (gp85, gH), BKRF2 (gp25, gL), or any variant thereof. As mentioned above, these EBV genes can also be classified as late-stage genes. In some embodiments, the polypeptide construct includes antigenic determinants derived from BLLF1 (gp350 / 220) or variant thereof. In some embodiments, the polypeptide construct includes antigenic determinants derived from BXLF2 (gp85, gH) or variant thereof. In some embodiments, the polypeptide construct includes antigenic determinants derived from BKRF2 (gp25, gL) or variant thereof.

[0085] In some embodiments, the polypeptide construct includes antigenic determinants derived from a single EBV antigen (such as an EBV polypeptide) or a variant thereof. For example, in some embodiments, the antigenic determinants in the polypeptide construct may be derived from a single EBV cleavage gene, a single EBV pre-latent gene, a single EBV latent gene, a single EBV early gene, a single EBV immediate early gene, a single EBV late gene, or any variant thereof. In some embodiments, the antigenic determinants in the polypeptide construct are derived from BLLF1 (gp350 / 220) or a variant thereof. In some embodiments, the antigenic determinants in the polypeptide construct are derived from BZLF1 or a variant thereof. In some embodiments, the antigenic determinants in the polypeptide construct are derived from LMP2B or a variant thereof.

[0086] In some embodiments, the antigenic determinants in the polypeptide construct are derived from at least two, at least three, at least four, at least five, at least six, or at least seven EBV antigens or variants thereof. In some embodiments, the antigenic determinants in the polypeptide construct are derived from two, three, four, five, six, or seven EBV antigens or variants thereof. In some embodiments, the antigenic determinants in the polypeptide construct are derived from fewer than two (e.g., one), fewer than three, fewer than four, fewer than five, fewer than six, or fewer than seven EBV antigens or variants thereof.

[0087] In some embodiments, the antigenic determinants in the polypeptide construct are derived from early EBV genes, latent EBV genes, and late EBV genes. In some embodiments, the antigenic determinants in the polypeptide construct are derived from EBV cleavage genes, latent EBV genes, and EBV latent-to-cleavage-reactivation genes. In some embodiments, the antigenic determinants in the polypeptide construct are derived from one or more EBV genes selected from the group consisting of BLLF1 (gp350 / 220), BZLF1, BXLF2 (gp85, gH), BKRF2 (gp25, gL), LMP2B, and combinations thereof. In some embodiments, the antigenic determinants in the polypeptide construct are derived from one or more EBV genes selected from the group consisting of BLLF1 (gp350 / 220), BZLF1, BXLF2 (gp85, gH), BKRF2 (gp25, gL), and LMP2B (see also Table 1). Without being bound by any particular theory, antigenic determinants derived from this gene combination are thought to enhance the biological activity of polypeptide constructs expressed from srRNA molecules. Specifically, antibodies against gp350 are believed to neutralize EBV entry into B cells, antibodies against gH are believed to neutralize EBV entry into B and / or epithelial cells, and antibodies against gL are generally believed to neutralize B cell and / or epithelial cell entry (involving EBV membrane / cell membrane fusion). Antibodies against LMP2B and BZLF1 are also thought to induce anti-EBV T cells, which can attack latent or reactivated lysed EBV-infected cells, respectively. Table 1 : A list of EBV antigens included in some exemplary srRNA constructs of this disclosure.

[0088] In some embodiments of this disclosure, the coding sequence of the polypeptide construct comprises the coding sequences of the following genes: (a) BZLF1 or a variant thereof; (b) BLLF1 (gp350 / 220) or a variant thereof; (c) LMP2B or a variant thereof; (d) gH and gL or variants thereof; (e) BZLF1, LMP2B, gH, gL, and BLLF1 (gp350 / 220) or any variant thereof; (f) gH, gL, and BLLF1 (gp350 / 220) or any variant thereof; or (g) BZLF1 and LMP2B or any variant thereof. In some embodiments, BLLF1 (gp350 / 220) or a variant thereof lacks a functional transmembrane (TM) domain.

[0089] In some embodiments, the peptide construct includes an antigenic determinant derived from BLLF1 (gp350 / 220) or a variant thereof, wherein BLLF1 (gp350 / 220) or a variant thereof lacks a functional transmembrane (TM) domain, for example, comprising one or more mutated and / or missing TM domains. Without being bound by any particular theory or mechanism of action, BLLF1 (gp350 / 220) lacking the TM domain is considered to exhibit a lower safety risk than full-length BLLF1 (gp350 / 220). Furthermore, transmembrane domains are generally not considered targets for neutralizing antibodies. Therefore, in some cases, removing the transmembrane domain from the peptide construct of this disclosure may help alleviate manufacturing-related size limitations. For example, in some embodiments, the peptide construct includes an antigenic determinant derived from BLLF1 (gp350 / 220) or a variant thereof, wherein at least a portion of the transmembrane (TM) domain has been deleted. In some embodiments, the TM domain of BLLF1 (gp350 / 220) or a variant thereof is completely absent.

[0090] In some embodiments, gH or its variants lack a functional transmembrane domain (TM), for example, containing one or more mutated and / or missing TM domains. Without being bound by any particular theory, gH lacking a TM domain is considered to exhibit a lower safety risk than full-length gH. Additionally, transmembrane domains are generally not considered targets for neutralizing antibodies. Therefore, in some cases, removing the transmembrane domain from the peptide construct of this disclosure helps mitigate size limitations associated with manufacturing. For example, in some embodiments, the peptide construct contains an antigenic determinant derived from gH or its variants, wherein at least a portion of the transmembrane (TM) domain has been deleted. In some embodiments, the TM domain of gH or its variants is completely absent.

[0091] In some embodiments, the BZLF1 or its variants lack (i) a functional nuclear localization sequence (NLS) and / or (ii) a functional transactivation (TA) domain. Without being bound by any particular theory, BZLF1 lacking both the NLS and TA domains is considered to exhibit a lower safety risk than full-length BZLF1. For example, in some embodiments, the peptide construct includes an antigenic determinant from BZLF1 or its variants, which contains one or more mutations and / or deletions in the NLS. In some embodiments, the peptide construct includes an antigenic determinant from BZLF1 or its variants, wherein at least a portion of the NLS has been deleted. In some embodiments, the NLS of the BZLF1 or its variants is completely absent.

[0092] In some embodiments, the peptide construct comprises an antigenic determinant derived from BZLF1 or a variant thereof, wherein BZLF1 or a variant thereof comprises one or more mutations and / or deletions in the transactivation (TA) domain. In some embodiments, the peptide construct comprises an antigenic determinant derived from BZLF1 or a variant thereof, wherein at least a portion of the TA domain has been deleted. In some embodiments, the TA domain of BZLF1 or a variant thereof is completely absent.

[0093] In some embodiments, the coding sequence of the polypeptide construct includes Figure 2The EBV antigen and antigen configuration and / or sequence described in [the original text]. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 1. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 2. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 4. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 5. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 6.In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 10.

[0094] In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 11. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 13. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 16. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 17. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18.In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 20.

[0095] In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 21. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 22. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 23. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 24. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 25. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 26. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 27. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28.In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 29. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 30.

[0096] In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 31. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 33. In some embodiments, the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 34.

[0097] In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 1, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 2, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 4, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 5, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 6, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 7, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 8, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids.In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 10, wherein one, two, three, four, five or more nucleotides of the amino acid sequence may be substituted with different amino acids.

[0098] In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 11, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 12, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 13, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 14, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 15, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 16, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 17, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 18, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 19, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 20, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids.

[0099] In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 21, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 22, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 23, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 24, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 25, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 26, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 27, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 28, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 29, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 30, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids.

[0100] In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 31, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 32, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 33, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 34, wherein one, two, three, four, five, or more nucleotides of said amino acid sequence may be substituted with different amino acids.

[0101] The nucleic acid constructs disclosed herein may or may not contain coding sequences for antigenic determinants derived from the EBNA1 gene. In some specific embodiments, the nucleic acid constructs disclosed herein do not contain coding sequences for antigenic determinants derived from the full-length EBNA1 gene. In some other embodiments, the nucleic acid constructs disclosed herein contain coding sequences for at least one antigenic determinant derived from the full-length EBNA1 gene.

[0102] The nucleic acid constructs disclosed herein may or may not contain coding sequences for antigenic determinants derived from the LMP1 gene and / or the EBNA1 gene. In some specific embodiments, the nucleic acid constructs disclosed herein do not include coding sequences for antigenic determinants derived from the full-length LMP1 gene. In some other embodiments, the nucleic acid constructs disclosed herein include at least one coding sequence for an antigenic determinant derived from the full-length LMP1 gene. In some specific embodiments, the nucleic acid constructs disclosed herein do not include coding sequences for antigenic determinants derived from the full-length LMP2A gene. In some other embodiments, the nucleic acid constructs disclosed herein include at least one coding sequence for an antigenic determinant derived from the full-length LMP2A gene.

[0103] As described above, in some embodiments of this disclosure, the nucleic acid construct comprises a nucleic acid sequence encoding a modified genome or srRNA of an alphavirus, wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of the modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct, the polypeptide construct containing one or more antigenic determinants derived from Epstein-Barr virus (EBV). In some embodiments, the modified alphavirus genome or srRNA lacks at least a portion of the nucleic acid sequence encoding one or more of the viral structural proteins CP, E1, E2, E3, and 6K in the unmodified alphavirus genome or srRNA. In some embodiments, the modified viral genome or srRNA lacks a partial or complete sequence encoding CP. In some embodiments, the modified viral genome or srRNA lacks a partial or complete sequence encoding E1. In some embodiments, the modified viral genome or srRNA lacks a partial or complete sequence encoding E2. In some embodiments, the modified viral genome or srRNA lacks a partial or complete sequence encoding E3. In some embodiments, the modified viral genome or srRNA lacks a partial or complete sequence encoding 6K. In some embodiments, the modified viral genome or srRNA lacks partial or complete sequences encoding a combination of CP, E1, E2, E3, and 6K. Therefore, some embodiments of this disclosure provide a modified alphavirus genome or srRNA containing coding sequences for the non-structural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified alphavirus genome or srRNA, but lacking at least partial or complete sequences encoding one or more structural proteins (such as CP, E1, E2, E3, and 6K) of the alphavirus genome or srRNA.

[0104] In some embodiments, the modified viral genome or srRNA lacks a majority of the nucleic acid sequence encoding one or more viral structural proteins. Those skilled in the art will understand that a majority of the nucleic acid sequence encoding a viral structural polypeptide may include a sufficient number of nucleic acid sequences encoding the viral structural polypeptide to provide a presumed identification of the polypeptide, either manually evaluated by those skilled in the art or by computer-automated sequence alignment and identification using algorithms such as the Basic Local Alignment Search tool (BLAST; publicly available, e.g., guides.lib.berkeley.edu / ncbi / blast and / / blast.ncbi.nlm.nih.gov / Blast.cgi). Thus, a majority of the nucleotide sequence contains sufficient sequence to provide specific identification and / or isolation of nucleic acid fragments containing said sequences. For example, a majority of the nucleic acid sequence may include at least about 20%, such as about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the full-length nucleic acid sequence. As described above, this disclosure provides nucleic acid molecules and constructs lacking partial or complete nucleic acid sequences encoding one or more viral structural proteins. Those skilled in the art who benefit from the sequences disclosed herein can readily use all or most of the sequences of this disclosure in the compositions and methods of this disclosure. Therefore, this application comprises the complete sequences disclosed herein, the sequences shown in the appended sequence listing, and most of the sequences as defined above.

[0105] In some embodiments, the modified viral genome or srRNA lacks the complete sequence encoding viral structural proteins; for example, the modified viral genome or srRNA does not include the nucleic acid sequence encoding the structural protein of the unmodified viral genome or srRNA.

[0106] In some embodiments, the nucleic acid constructs disclosed herein further include one or more expression cassettes, for example, for expressing a peptide construct of interest, such as a peptide construct containing one or more antigenic determinants derived from Epstein-Barr virus (EBV). In principle, the nucleic acid constructs disclosed herein can generally contain any number of expression cassettes. In some embodiments, the nucleic acid constructs disclosed herein may contain at least two, at least three, at least four, at least five, or at least six expression cassettes. Those skilled in the art will understand that the term "expression cassette" generally refers to a construct of genetic material containing a coding sequence and sufficient regulatory information to guide the correct transcription and / or translation of the coding sequence in vivo and / or in vitro cells. Expression cassettes may be inserted into vectors to target desired host cells and / or be inserted into subjects. Therefore, in some embodiments, the term "expression cassette" may be used interchangeably with the term "expression construct." In some embodiments, the term "expression cassette" refers to a nucleic acid construct comprising a gene or functional RNA encoding a protein operatively linked to regulatory elements (e.g., promoters and / or termination signals, and optionally, any other nucleic acid sequence or combination of other nucleic acid sequences affecting the transcription or translation of the gene).

[0107] In some embodiments, at least one of the expression cassettes includes a promoter operatively linked to a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequence may encode a polypeptide construct of interest, for example, a polypeptide construct containing one or more antigenic determinants derived from Epstein-Barr virus (EBV). Therefore, nucleic acid constructs as provided herein can be applied, for example, as expression vectors, to influence the expression of heterologous nucleic acid sequences (such as heterologous nucleic acid sequences encoding polypeptide constructs containing one or more antigenic determinants derived from Epstein-Barr virus (EBV)) when including regulatory elements (such as promoters) operatively linked to the heterologous nucleic acid sequence. Both naturally occurring promoter sequences and artificially synthesized (and / or generated) promoter sequences are suitable. Therefore, in some embodiments, the promoter is or contains a naturally occurring sequence of a genomic sequence isolated from or derived from a gene. In some embodiments, the promoter can be synthetically generated or designed by altering known DNA elements. In some embodiments, at least one of the expression cassettes includes a subgenomic (sg) promoter operatively linked to a heterologous nucleic acid sequence. In some embodiments, the sg promoter sequence is a 26S subgenomic promoter. In some embodiments, at least one non-structural protein (nsP) or a portion thereof of the modified alphavirus genome or srRNA is heterologous relative to the remainder of the modified alphavirus genome or srRNA. In some embodiments, the modified alphavirus genome or srRNA further comprises a nucleic acid sequence encoding a heterologous nsP or a portion thereof. In some embodiments, the nucleic acid molecule of this disclosure further comprises one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR. For example, in some embodiments, the nucleic acid molecule of this disclosure may include a native 5' UTR and a heterologous 3' UTR. In some embodiments, the nucleic acid molecule of this disclosure may include a heterologous 5' UTR and a native 3' UTR. In some embodiments, both the 5' UTR and the 3' UTR are heterologous relative to the remainder of the modified alphavirus genome or srRNA.

[0108] In some embodiments, at least one of the expression cassettes contains the coding sequence of a gene of interest (GOI). In some embodiments, the coding sequence of the GOI contains the coding sequence of a polypeptide construct of interest (PCI). In some embodiments, the polypeptide construct of interest contains a single EBV polypeptide sequence (e.g., a single-gene PCI). In some embodiments, the coding sequence of the PCI contains coding sequences of multiple EBV polypeptides, such as multi-gene PCIs (e.g., dual-gene, triple-gene, or quadruple-gene, etc.). In some embodiments, each of the multiple polypeptide coding sequences is operatively linked to a separate promoter sequence. In some embodiments, the coding sequences of the multiple polypeptides are operatively linked to each other within a single open reading frame, for example, in a polycistronic ORF (see, for example, Example 1, Rep-674, Rep-675, Rep-678, Rep-679, Rep-680, Rep-695, Rep-728, Rep-729, Rep-730, Rep-731, and Rep-733). In some embodiments, the coding sequences of the plurality of polypeptides are configured in two or more ORFs, which are linked to each other by sequences of internal ribosome entry sites (IRES) (see, for example, Example 1, Rep-692, Rep-693, Rep-694, Rep-696, Rep-697, Rep-698, Rep-699, Rep-734, Rep-735, Rep-736 and Rep-737).

[0109] In some embodiments, the coding sequences of one or more polycistronic ORFs are operatively linked to a promoter sequence. In some embodiments, at least one promoter sequence is a subgenomic (sg) promoter. In some embodiments, the sg promoter is a 26S genomic promoter.

[0110] In some embodiments, multiple EBV peptides may be linked to each other directly or indirectly (e.g., via one or more linker sequences, such as adapters). For example, in some embodiments, the multiple EBV peptides may be directly linked to each other, such as being adjacent to each other. In some embodiments, at least two (e.g., 2, 3, 4, or 5) of the multiple EBV peptides are operatively linked to each other via one or more linker sequences (e.g., adapters). In some embodiments, the length and amino acid composition of the linker sequences may be optimized to alter the orientation, flexibility, and / or proximity of the peptides relative to each other to achieve a desired PCI activity or property. In some embodiments, one of the multiple linker sequences (e.g., adapters) comprises one or more autologous proteolytic peptide sequences. Non-limiting examples of autologous proteolytic peptide sequences suitable for the methods and compositions of this disclosure include those derived from calcium-dependent serine endonuclease (furin), porcine cephalovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis virus A (ERAV) 2A (E2A), *Typha laniceps* virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), and muscular dystrophy virus 2A (BmIFV2A). In some embodiments, the polypeptide constructs of this disclosure comprise one or more P2A autologous proteolytic cleavage sequences. In some embodiments, at least two of the plurality of polypeptides are operatively linked to each other via P2A autologous proteolytic cleavage sequences.

[0111] In some embodiments, the coding sequences of the plurality of polypeptides and their arrangement are configured as follows: Figure 2 As shown. In some embodiments, the encoded sequence of the PCI includes encoded sequences of (i) gH, (ii) gL, or any variant thereof. In some embodiments, the encoded sequences of (i) gH, (ii) gL, or any variant thereof are operatively connected to each other within a single polycistronic ORF.

[0112] In some embodiments, the encoded sequence of the PCI includes encoded sequences of (i) gH, (ii) gL, (iii) gp350, or any variant thereof. In some embodiments, the encoded sequences of (i) gH, (ii) gL, (iii) gp350, or any variant thereof are operatively concatenated with each other within a single polycistronic ORF.

[0113] In some embodiments, the encoded sequence of the PCI includes encoded sequences of (i) BZLF1, (ii) gH, (iii) gL, (iv) gp350, and (v) LMP2B, or any variant thereof. In some embodiments, the encoded sequences of (i) BZLF1, (ii) gH, (iii) gL, (iv) gp350, and (v) LMP2B, or any variant thereof, are operatively concatenated with each other within a single polycistronic ORF.

[0114] In some embodiments, the encoded sequences of (i) BZLF1, (ii) gH, (iii) gL, (iv) gp350, and (v) LMP2B, or any variant thereof, are configured into a first ORF and a second ORF connected to each other via IRES. The use of the terms "first," "second," "third," or "fourth" ORF in the encoded sequences of the PCI herein is not intended to imply any particular structural arrangement of the "first," "second," "third," or "fourth" ORFs in the PCI. In some embodiments, the first ORF contains the encoded sequence of BZLF1, or a variant thereof; and the second ORF contains the encoded sequences of (i) gH, (ii) gL, (iii) gp350, and (iv) LMP2B, or any variant thereof. In some embodiments, the first ORF contains the encoded sequences of (i) BZLF1 and (ii) gH and (iii) gL, or any variant thereof; and the second ORF contains the encoded sequences of (i) gp350 and (ii) LMP2B, or any variant thereof.

[0115] In some implementations, the first ORF contains encoding sequences of (i) BZLF1, (ii) gH and (iii) gL or any variant thereof; and the second ORF contains encoding sequences of (i) gp350 and (ii) LMP2B or any variant thereof.

[0116] In some implementations, the first ORF contains encoding sequences of (i) BZLF1, (ii) gH, (iii) gL and (iv) gp350 or any variant thereof; and the second ORF contains encoding sequences of LMP2B or variant thereof.

[0117] In some embodiments, the first ORF contains encoded sequences of (i) gH, (ii) gL, and (iii) gp350 or any variant thereof; and the second ORF contains encoded sequences of (i) BZLF1 and (ii) LMP2B or any variant thereof. In some embodiments, the first ORF contains encoded sequences of (i) gH, (ii) gL, and (iii) gp350 or any variant thereof; and the second ORF contains encoded sequences of (i) LMP2B and (ii) BZLF1 or any variant thereof. In some embodiments, the first ORF contains encoded sequences of (i) gH, (ii) gL, (iii) gp350, and (iv) LMP2 or any variant thereof; and the second ORF contains encoded sequences of BZLF1 or any variant thereof.

[0118] In some implementations, the first ORF contains encoding sequences of (i) gp350, (ii) gH, (iii) gL and (iv) LMP2B or any variant thereof; and the second ORF contains encoding sequences of BZLF1 or any variant thereof.

[0119] In some embodiments, the first ORF contains an encoded sequence of gp350 or a variant thereof; and the second ORF contains encoded sequences of (i) gH and (ii) gL or any variant thereof. In some embodiments, the first ORF contains encoded sequences of (i) gL and (ii) gH or any variant thereof; and the second ORF contains encoded sequences of gp350 or a variant thereof. In some embodiments, the first ORF contains an encoded sequence of gp350 or a variant thereof; and the second ORF contains encoded sequences of (i) gL and (ii) gH or any variant thereof.

[0120] In some embodiments, the first ORF contains an encoding sequence of LMP2B or a variant thereof; and the second ORF contains an encoding sequence of BZLF1 or a variant thereof.

[0121] In some embodiments, the gp350 and / or gH sequences lack a functional transmembrane domain (-TM). In some embodiments, the BZLF1 sequence lacks (i) a functional nuclear localization sequence (NLS) and (ii) a functional transactivation (TA) domain.

[0122] There are no particular limitations on the EBV strains suitable for the compositions and methods of this disclosure. The EBV strains used in the compositions and methods disclosed herein can be any EBV strain. Both toxic and non-toxic EBV strains are suitable. In some embodiments, one or more EBV antigenic determinants are type 1 EBV (EBV-1) or type 2 EBV (EBV-2) or combinations thereof. Non-limiting examples of EBV strains suitable for the compositions and methods of this disclosure include the AG876 strain, Akata strain, Alaska strain, B95-8 strain, Hina 1 strain, HKNPC strain, China 2 strain, CVI988 strain, GD1 strain, GD2 strain, GP202 strain, M81 strain, Mediterranean strain, Mutu strain, P3HR1 strain, Raji strain, SNU-719 strain, YCCEL1 strain, or combinations thereof.

[0123] In some embodiments, the coding sequences of the plurality of polypeptides are operatively linked to each other via one or more internal ribosome entry sites (IRES). Non-limiting examples of IRES suitable for the methods and compositions of this disclosure include viral IRES sequences, cellular IRES sequences, and artificial IRES sequences. Examples of suitable IRES include, but are not limited to, Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, cyprinivirus IRES, cricket paralysis virus IRES, aphid virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, microRNA virus IRES, encephalomyelitis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES. In some embodiments, the internal ribosome entry site (IRES) is derived from encephalomyelitis virus (EMCV). In some embodiments, the one or more IRES sequences comprise the sequence of SEQ ID NO: 58.

[0124] In some embodiments of the methods described herein, the recombinant alphavirus srRNA is a virus belonging to the genus Alphavirus of the family Togaviridae. In some embodiments of this disclosure, the modified alphavirus genome or srRNA is an alphavirus belonging to the Venezuelan equine encephalitis virus / Eastern equine encephalitis virus (VEEV / EEEV) group, the Semliki Forest Virus (SFV) group, or the Simbis Virus (SINV) group. In some embodiments, the modified alphavirus genome or srRNA is an alphavirus belonging to the BFV complex, EEEV complex, MIDV complex, NDUV complex, SFV complex, VEEV complex, or WEEV complex. In some implementations, the A virus is Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Swamp Virus (EVEV), Mucombo Virus (MUCV), Picsuna Virus (PIXV), Middleburg Virus (MIDV), Chikungunya Virus (CHIKV), Orongrong Virus (ONNV), Ross River Virus (RRV), Bama Forest Virus (BF), Geta Virus (GET), SAGV, Bibaru Virus (BEBV), Mayaro Virus (MAYV), UNAV, SINV, AURAV, WHAV, Babanki Virus (BABV), Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus (WEEV), Highland J Virus (HJV), Morganburg Virus (FMV), Ndumu Virus (NDUV), Madariga Virus (MADV), or Worm Creek Virus. In some embodiments, the alphavirus is VEEV, EEEV, CHIKV, or SINV. In some embodiments, the alphavirus is VEEV. In some embodiments, the alphavirus is EEEV. In some embodiments, the alphavirus is Western equine encephalitis virus (WEEV). In some embodiments, the alphavirus is CHIKV. In some embodiments, the alphavirus is SINV.

[0125] In some embodiments, the alphavirus is chikungunya virus (CHIKV). Non-limiting examples of CHIKV strains suitable for the compositions and methods of this disclosure include CHIKV S27, CHIKV LR2006-OPY-1, CHIKVYO123223, CHIKV DRDE, CHIKV 37997, CHIKV 99653, CHIKV Ag41855, and Nagpur (India) 653496 strain. Both toxic and non-toxic CHIKV strains are applicable. Further examples of CHIKV strains suitable for the compositions and methods of this disclosure include, but are not limited to, Afreen et al., Immunol. 2014, 58:688-696, Lanciotti and Lambert ASTMH 2016, 94(4):800-803, and Langsjoen et al., 2018, 9(2):e02449-17. In some embodiments, the modified CHIKV genomic or replicon RNA (e.g., self-replicating RNA) is derived from CHIKV strain S27. In some embodiments, the modified CHIKV genomic or replicon RNA is derived from CHIKV strain DRDE. In some embodiments, the modified CHIKV genomic or replicon RNA is derived from CHIKV strain DRDE-06. In some embodiments, the modified CHIKV genomic or replicon RNA is derived from CHIKV strain DRDE-07. In some implementations, the modified CHIKV genome or replicon RNA is derived from the CHIKV strain S27.

[0126] In some embodiments, the alphavirus is Eastern Equine Encephalitis Virus (EEEV). Non-limiting examples of EEEV strains suitable for the compositions and methods of this disclosure include EEEV 792138, 783372, BeAn5122, BeAr300851, BeAr436087, C-49, FL91-4679, FL93-939, GML903836, MP-9, PE6, and V105-00210. Both virulent and non-virulent EEEV strains are suitable. Other suitable EEEV strains include, but are not limited to, those described on the Virus Pathogen Resource Website (ViPR; publicly available at www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=868&decorator=toga). In some embodiments, the modified EEEV genome or replicon RNA (e.g., self-replicating RNA) is derived from EEEV strain FL93-939.

[0127] In some embodiments, the alphavirus is Sindbis virus (SINV). In some embodiments, the modified genome or RNA replicon (such as self-replicating RNA) is a SINV strain. Non-limiting examples of SINV strains suitable for the compositions and methods of this disclosure include SINV strains AR339, AR86, and Girdwood. Examples of SINV strains suitable for the compositions and methods of this disclosure include, but are not limited to, Sammels et al. J. Gen. Virol. 1999, 80(3):739-748, Lundström and Pfeffer Vector Borne Zoonotic Dis. 2010, 10(9):889-907, Sigei et al. Arch. of Virol. 2018, 163:2465-2469, and Ling et al. J. Virol. 2019, 93:e00620-19. Other suitable SINV strains include, but are not limited to, those described in the Virus Pathogen Resource website (ViPR; publicly available at www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=868&decorator=toga). Both virulent and non-virulent SINV strains are suitable. In some embodiments, the modified genome or RNA replicon is from the SINV strain Girdwood. In some embodiments, the modified genome or RNA replicon is from the SINV strain AR86. In some embodiments, the modified SINV genome or replicon RNA is derived from the SINV strain Girdwood. In some embodiments, the modified SINV genome or replicon RNA is derived from the SINV strain AR86. In some embodiments, at least one heterologous nsP or a portion thereof of the modified genome or RNA replicon is derived from the SINV strain AR86. In some embodiments, the at least one heterologous nsP or a portion thereof is nsP1, nsP3, nsP4, or any portion thereof, or any combination thereof. In some implementations, the modified genome or RNA replicon is of SINV strain AR86.

[0128] In some embodiments, the alphavirus is Western Equine Encephalitis Virus (WEEV). Non-limiting examples of WEEV strains suitable for the compositions and methods of this disclosure include WEEV California, McMillan, IMP181, Imperial, Imperial 181, IMPR441, 71V-1658, AG80-646, BFS932, COA592, EP-6, E1416, BFS1703, BFS2005, BSF3060, BSF09997, CHLV53, KERN5547, 85452NM, Montana-64, S8-122, and TBT-235. Further examples of WEEV strains suitable for the compositions and methods of this disclosure include 5614, 93A27, 93A30, 93A38, 93A79, B628 (Cl 15), CBA87, CNTR34, CO921356, Fleming, Lake43, PV012357A, PV02808A, PV72102, R02PV001807A, R02PV002957B, R02PV003422B, R05PV003422B, R0PV003814A, and R0PV00384A. Both toxic and non-toxic WEEV strains are applicable. Other suitable WEEV strains include, but are not limited to, those described in Bergren NA et al., J. Virol. 88(16):9260-9267, August 2014, and the Virus Pathogen Resource website (ViPR; publicly available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=57240&decorator=toga). In some embodiments, the modified WEEV genome or srRNA is derived from the WEEV strain Imperial. In some embodiments, the modified WEEV genome or srRNA is derived from the WEEV strain McMillan.

[0129] In some embodiments, the alphavirus is Madariga virus (MADV), formerly known as East South American equine encephalitis virus (SA EEEV). Non-limiting examples of MADV strains suitable for the compositions and methods of this disclosure include ArgLL, ArgB, BeAn-5122, ArgM, 24443 (TR59), 25714 (BG60), BeAr 18205, 900188 (PA62), BeAr 81828, BeAr 126650, 68U231, 77U1104 (PE70), 75V1496, BeAr 300851, 75U40, and ElDelirio (Arrigo NC et al., ibid., 2010). Further examples of MADV strains suitable for the compositions and methods of this disclosure include 76V25343, 77U1 (BR77), BeAr348998, IVICPan57151, BeAn416361, 903836 (PA84), BeAr436087, 435731 (PA86), C49 (CO92), PE-0.0155-96 (0.0155), PE-3.0815-96 (3.0815), PE-16.0050-98 (16.0050), PE-18.0140-99 (18.0140), and PE-18.0172-99 (18.0172) (Arrigo NC et al., ibid., 2010). Other suitable MADV strains include, but are not limited to, those described by Arrigo NC et al., ibid., 2010, and those publicly available on the Virus Pathogen Resource website (ViPR; publicly available at www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=868&decorator=toga). In some embodiments, the modified MADV genome or srRNA is derived from the MADV strain BeAr300851.

[0130] In some embodiments, the srRNA construct of this disclosure may include a 5' cap. As used herein, the term "5' cap" refers to the 5' end structure of some eukaryotic RNAs (such as RNA transcripts) and typically contains a dinucleotide or guanylic acid nucleotide linked to RNA (such as mRNA) via a 5'-to-5'-triphosphate bond (also known as Gppp or G(5')ppp(5')). In some embodiments, the guanosine included in the 5' cap may be modified, for example, by methylation at one or more sites (e.g., position 7) of the base (guanine), and / or by methylation at one or more sites of the ribose. In some embodiments, the guanosine included in the 5' cap includes 2'O methylation at the ribose (2'OMeG). In some embodiments, the guanosine included in the 5' cap includes methylation at position 7 of guanine (m7G). In some embodiments, the guanosine nucleotide included in the 5' cap comprises methylation at the 7-position of guanine and 2'O methylation at the ribose (m7(2'OMeG)).

[0131] A variety of different cap structures can be used to generate 5′ caps for srRNA synthesized through in vitro transcription. In some embodiments, srRNAs having a 5′ cap or a 5′ cap analog disclosed herein can be provided via in vitro transcription, wherein the 5′ cap is co-transcribed into the RNA strand, or can be attached to the RNA post-transcriptionally using a capping enzyme. Thus, in some embodiments, the 5′ capping of the synthesized srRNA can be co-transcribed with a chemical cap analog (i.e., capping during in vitro transcription). For example, the CleanCap® technology uses commercially available reagents and AG initiators in co-transcriptional reactions to provide highly efficient capping (90%+) to provide a native Cap 1 structure with 2′-O-methyl and N7-methyl groups on different guanine components. As another example, the antiretroviral cap analog (ARCA) cap contains a 5′-5′-guanine triphosphate-guanine bond, one of which contains an N7-methyl group and a 2′-O-methyl group. Alternatively, in some embodiments, the synthesized srRNA molecule can also be enzymatically capped post-transcriptionally. These may generate more realistic 5' cap structures, structurally or functionally closer to the endogenous 5' cap, which enhances the binding of cap-binding proteins, prolongs the half-life, reduces susceptibility to 5' endonucleases, and / or reduces 5' uncapping. Many synthetic 5' cap analogues have been developed and are known in the art for enhancing mRNA stability and translatability (see, for example, Grudzien-Nogalska E. et al., Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0132] Therefore, in some embodiments, the srRNA disclosed herein is a co-transcribed capped srRNA. Exemplary co-transcribed capped srRNAs include, but are not limited to, antireverse cap analogs (ARCA) and CleanCap® srRNA.

[0133] In other embodiments, the srRNA disclosed herein is an enzymatically capped srRNA. Exemplary capping enzymes include, but are not limited to, vaccinia virus capping enzyme (VCE) and Faustovirus capping enzyme (FCE).

[0134] In some embodiments, the modified alphavirus genome or srRNA is an alphavirus belonging to the VEEV / EEEV group, or the SFV group, or the SINV group. In some embodiments, the alphavirus is Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Chikungunya virus (CHIKV), Western equine encephalitis virus (WEEV), or Sindbis virus (SINV).

[0135] In some embodiments, the coding sequence of the polypeptide construct of interest (PCI) is redesigned and / or optimized to obtain desired properties, such as increased stability, potency, and expression (e.g., translation efficiency), which in turn can maximize the impact of the production, delivery, and administration of the biotherapeutic agent. For example, in some embodiments, the coding sequence of the PCI is optimized to express at a level higher than that of a reference coding sequence. Regarding sequence optimization of nucleotide sequences, the degeneracy of the genetic code provides the possibility of substituting at least one base of the gene sequence encoding the protein with a different base without altering the amino acid sequence of the polypeptide produced by said gene. Thus, nucleic acid constructs of this disclosure may also have any base sequence altered by substituting any polynucleotide sequence disclosed herein, based on the degeneracy of the genetic code. References describing codon usage are readily available from the public. In some embodiments, polynucleotide sequence variants may be generated for a variety of reasons, such as to optimize expression in a particular host (e.g., changing the codon usage in alphavirus mRNA to those preferred by other organisms (e.g., humans, non-human primates, hamsters, mice, or monkeys). Therefore, in some implementations, the coding sequence of PCI is optimized for expression in target host cells using codons optimized for expression. The technique for constructing a synthetic nucleic acid sequence encoding PCI using preferred codons most suitable for host cell expression can be determined by computational methods that analyze the commonalities and relative abundance of codon usages encoding native proteins in the host cell genome using techniques well-known in the art. Codon usage databases (http: / / www.kazusa.or.jp / codon) can be used to generate codon-optimized sequences for mammalian cellular environments. Additionally, various software tools can be used to convert sequences from one organism into optimal codons for different host organisms, such as the JCat codon optimization tool (www.jcat.de), the Integrated DNA Technology (IDT) codon optimization tool (https: / / www.idtdna.com / CodonOpt), or the online codon optimization tool Optimizer (http: / / genomes.urv.es / OPTIMIZER). Such synthetic sequences can be constructed using techniques known in the art for constructing synthetic nucleic acid molecules and are available from various commercial vendors. Therefore, in some embodiments, the coding sequence of PCI is optimized for expression at a higher level than that of a reference coding sequence (e.g., an uncodontated coding sequence). In some embodiments, the codont-optimized sequence of PCI results in an increase in expression level of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to an uncodontated reference coding sequence.In some implementations, the codon-optimized sequence of PCI results in an expression level increase of at least 2, 3, 4, or 5 times compared to the uncodon-optimized reference coding sequence.

[0136] In some implementations, the coding sequence of PCI is optimized to enhance RNA stability and / or expression. RNA stability is generally associated with the RNA's "half-life." "Half-life" refers to the time period required to eliminate half of the activity, amount, or number of molecules. In the context of this disclosure, the half-life of RNA indicates the stability of the RNA. The half-life of RNA can affect the "expression duration" of the RNA. More information on principles, strategies, and methods for enhancing RNA stability can be found in Leppek K. et al., Nat. Commun. 13, 1536 (2022).

[0137] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding a polypeptide construct containing one or more antigenic determinants derived from Epstein-Barr virus (EBV), and the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35-56.

[0138] In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 36. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 37. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 41. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 42. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 43.In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 45. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 46.

[0139] In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 47. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 48. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 49. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 50. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 54. In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 55.In some embodiments, the nucleic acid sequence encoding the polypeptide construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 56.

[0140] In some embodiments, the nucleic acid construct of this disclosure includes a nucleic acid sequence encoding a polypeptide construct, said polypeptide construct containing one or more antigenic determinants derived from Epstein-Barr virus (EBV), and said nucleic acid sequence having 100% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35-56, wherein one, two, three, four, five or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure includes a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 35, wherein one, two, three, four, five or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure includes a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 36, wherein one, two, three, four, five or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 37, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 38, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 39, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 40, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 41, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 42, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 43, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides.In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 44, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 45, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 46, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides.

[0141] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 47, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 48, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 49, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 50, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 51, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 52, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 53, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 54, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 55, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 56, wherein one, two, three, four, five, or more nucleotides of said nucleic acid sequence may be substituted with different nucleotides.

[0142] Nucleic acid sequences having high sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with sequences selected from the group consisting of SEQ ID NO: 35-56 can be identified and / or isolated by viral genome sequence analysis, hybridization, and / or PCR using degenerate primers or gene-specific primers from the sequences identified in the corresponding SEQ ID NOS, using sequences identified herein (e.g., SEQ ID NO: 35-56) or any other sequences known in the art.

[0143] The molecular techniques and methods for assembling and characterizing these novel nucleic acid constructs are described in more detail in the embodiments of this application. In some embodiments, the nucleic acid molecules disclosed herein can be generated by DNA recombination techniques (such as polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. As disclosed herein, nucleic acid molecules include natural nucleic acid molecules and their homologs, including but not limited to natural allelic variants and modified nucleic acid molecules in which one or more nucleotide residues are inserted, deleted, and / or substituted, such modifications being performed in a manner that provides the desired properties to achieve the biological activities described herein. Therefore, in some embodiments, the nucleic acid molecules are recombinant nucleic acid molecules.

[0144] Those skilled in the art will understand that nucleic acid molecules (containing variants of naturally occurring nucleic acid sequences) can be produced using a variety of methods known to those skilled in the art (see, for example, Sambrook et al., In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). The sequence of a nucleic acid molecule can be modified relative to its naturally occurring sequence using various techniques, including but not limited to classical mutagenesis and recombinant DNA techniques, such as, but not limited to, site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction endonuclease cleavage of nucleic acid fragments, ligation of nucleic acid fragments, PCR amplification and / or mutagenesis of selected regions of nucleic acid sequences, recombinant cloning, and chemical synthesis, including the chemical synthesis of oligonucleotide mixtures and the ligation of mixture groups to “build” mixtures and combinations thereof of nucleic acid molecules. Homologous nucleic acid molecules can be selected from a mixture of modified nucleic acid molecules by screening for the function of proteins or srRNA encoded by nucleic acid molecules and / or by hybridization with wild-type genes or fragments thereof, or by using primer PCR with homology to a target or wild-type nucleic acid molecule or sequence. B. Recombinant cells

[0145] The nucleic acid constructs of this disclosure can be introduced into host cells to produce recombinant cells containing the nucleic acid molecules described herein. Therefore, prokaryotic or eukaryotic cells containing nucleic acid constructs encoding the modified alphavirus genome as described herein are also characteristic of this disclosure. In related aspects, some embodiments disclosed herein involve introducing nucleic acid constructs as provided herein into host cells (e.g., animal cells), followed by selection or screening of transformed cells and / or transgenic animals. The introduction of the nucleic acid constructs of this disclosure into cells and / or animals can be performed by methods known to those skilled in the art, such as, for example, viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, nuclear transfection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0146] Therefore, some embodiments of this disclosure relate to recombinant cells, such as recombinant animal cells containing nucleic acid constructs as described herein. The nucleic acid constructs may be stably integrated into the host genome, or may be replicated as episomes, or may exist in the recombinant host cell as microcircular expression vectors for stable or transient expression. Thus, in some embodiments of this disclosure, the nucleic acid constructs are maintained and replicated as episome units in the recombinant host cell. In some embodiments, the nucleic acid constructs are stably integrated into the genome of the recombinant cell. Stable integration can be accomplished using classical random genomic recombination techniques or more precise genome editing techniques (such as CRISPR / Cas9 or TALEN genome editing guided by guide RNA). In some embodiments, the nucleic acid molecule exists in the recombinant host cell as a microcircular expression vector for stable or transient expression. In some embodiments, the recombinant cells and / or transgenic animals contain srRNA molecules as described herein, for example, RNA molecules containing all the genetic information required to direct self-amplification or self-replication within the cell and / or animal.

[0147] In some embodiments, the recombinant cells are prokaryotic cells (such as the bacteria *Escherichia coli* (E. coli)) or eukaryotic cells (such as insect cells (e.g., mosquito cells or Sf21 cells) or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells)). In some embodiments, the cells are in vivo. In some embodiments, the cells are ex vivo. In some embodiments, the cells are in vitro. In some embodiments, the recombinant cells are eukaryotic cells. In some embodiments, the recombinant cells are animal cells. In some embodiments, the animal cells are vertebrate cells or invertebrate cells. In some embodiments, the recombinant cells are mammalian cells. Non-limiting examples of recombinant cells suitable for the methods and compositions of this disclosure include monkey kidney CV1 cells (COS-7) transformed with SV40, human embryonic kidney cells (e.g., HEK 293 or HEK 293 cells) or derived cells thereof (e.g., BHK-21 or BHK-570 cells), young hamster kidney cells (BHK), mouse Support cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 cells), human cervical cancer cells (e.g., HeLa cells), canine kidney cells (MDCK cells), buffalo rat hepatocytes (e.g., BRL 3A cells), human lung cells (e.g., W138 cells), and human hepatocytes (e.g., Hep cells). G2 cells), mouse mammary tumors (e.g., MMT060562 cells), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (e.g., Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NSO mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophages, human RAW 264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells. In some embodiments, recombinant cells are cells derived from the above-mentioned cells (i.e., derived cells of the original cells described herein), such as, for example, cells expanded from clones of the original cells, engineered versions of the original cells, or reclassified original cells after extensive passage or passage through another host.

[0148] In some embodiments, the recombinant cells are insect cells, such as cells from an insect cell line. In some embodiments, the recombinant cells are Sf21 cells. Other suitable insect cell lines include, but are not limited to, cell lines established from the orders Diptera, Lepidoptera, and Hemiptera, and may be derived from different tissue sources. In some embodiments, the recombinant cells are cells from a Lepidoptera cell line. The availability of such Lepidoptera insect cell lines has increased by approximately 50 lines per decade over the past few decades. More information on available Lepidoptera insect cell lines can be found, for example, in Lynn DE, Available lepidopteran insect celllines. Methods Mol Biol. 2007;388:117-38, which is incorporated herein by reference. In some embodiments, the recombinant cells are mosquito cells, for example, cells of mosquito species within the genera Anopheles, Culex, and Aedes (subgenus Stegomyia) (Ae.). Exemplary mosquito cell lines suitable for the compositions and methods described herein comprise cell lines from the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes tristralate, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culextritaeniorhynchus, Culex bitaeniorhynchus, and Toxorhynchites amboinensis. Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP-61, At GRIP-1, At GRIP-2, UM-AVE1, Mos.55, Sua1B, 4a-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cells are cells from the C6 / 26 cell line.

[0149] On the other hand, this document provides cell cultures comprising at least one recombinant cell as disclosed herein and a culture medium. Typically, the culture medium can be any suitable medium for culturing the cells described herein. Techniques for transforming a wide variety of the aforementioned host cells and species are known in the art and described in the technical and scientific literature. Therefore, cell cultures comprising at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for producing and maintaining cell cultures are known in the art. C. Genetically modified animals

[0150] On the other hand, transgenic animals comprising nucleic acid constructs as described herein are also provided. In some embodiments, the transgenic animal is a vertebrate or invertebrate. In some embodiments, the transgenic animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the transgenic animal is a mammal. In some embodiments, the transgenic mammal is a non-human mammal. In some embodiments, the transgenic animal produces polypeptide constructs, EBV antigens, and / or EBV antigenic determinants as described herein.

[0151] The transgenic non-human host animals disclosed herein are prepared using standard methods known in the art for introducing exogenous nucleic acids into the genome of non-human animals. In some embodiments, the non-human animals of this disclosure are non-human primates. Other animal species suitable for the compositions and methods of this disclosure include: (i) animals suitable for transgenic preparation; and (ii) animals capable of rearranging immunoglobulin gene segments to produce an antibody response. Examples of such species include, but are not limited to, mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cattle. Routes and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include prokaryotic microinjection, DNA microinjection, lentiviral vector-mediated DNA transfer to early embryos and sperm-mediated transgenic preparation, adenovirus-mediated DNA introduction into animal sperm (e.g., in pigs), retroviral vectors (e.g., in avian species), and somatic cell nuclear transfer (e.g., in goats). Prior art for the preparation of transgenic livestock animals is reviewed in Niemann, H. et al. (2005), Rev. Sci. Tech. 24:285-298.

[0152] In some embodiments, the animal is a vertebrate or invertebrate. In some embodiments, the animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the animal is a mammalian subject. In some embodiments, the mammal is a non-human animal. In some embodiments, the mammal is a non-human primate. In some embodiments, the transgenic animals of this disclosure can be produced using classical random genomic recombination techniques or using more precise techniques such as guide RNA-guided CRISPR / Cas genome editing, DNA-guided endonuclease genome editing using NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nuclease). In some embodiments, the transgenic animals of this disclosure can be produced using transgenic microinjection techniques and do not require the use of homologous recombination techniques, and are therefore considered easier to prepare and select than methods using homologous recombination. D. Pharmaceutical Composition

[0153] The nucleic acid constructs and / or recombinant cells disclosed herein can be incorporated into compositions (including pharmaceutical compositions). Such compositions typically comprise one or more nucleic acid constructs and / or recombinant cells described and provided herein, along with pharmaceutically acceptable excipients, such as carriers. In some embodiments, the compositions of this disclosure are formulated for the prevention, treatment, or management of health conditions, such as EBV-related diseases or infections (e.g., EBV infection). For example, the compositions of this disclosure can be formulated as prophylactic compositions, therapeutic compositions, or pharmaceutical compositions or mixtures thereof comprising pharmaceutically acceptable excipients. In some embodiments, the compositions of this disclosure are formulated for use as vaccines. In some embodiments, the compositions of this disclosure are formulated for use as adjuvants.

[0154] Therefore, in one respect, this document provides pharmaceutical compositions comprising pharmaceutically acceptable excipients and: (a) nucleic acid constructs of this disclosure, and / or (b) recombinant cells of this disclosure.

[0155] Non-limiting exemplary embodiments of the pharmaceutical compositions disclosed herein may include one or more of the following features. In some embodiments, compositions comprising nucleic acid constructs as disclosed herein and pharmaceutically acceptable excipients are provided. In some embodiments, compositions comprising recombinant cells as disclosed herein and pharmaceutically acceptable excipients are provided.

[0156] In some embodiments, the nucleic acid constructs of this disclosure (such as vectors or srRNA molecules) may be used in naked form or formulated with a delivery medium. Exemplary delivery media suitable for the compositions and methods of this disclosure include, but are not limited to, exosomes, liposomes (e.g., neutral or anionic liposomes), microspheres, immunostimulatory complexes (ISCOMS), lipid-based nanoparticles (LNPs), solid lipid nanoparticles (SLNs), polymers, polymer nanoparticles, viral replicon particles (VRPs), or conjugated with bioactive ligands, which may facilitate delivery and / or enhance immune responses against EBV. These compounds are readily available to those skilled in the art; see, for example, Liposomes: A Practical Approach, RCPNew Ed, IRL press (1990) and Mendes BB et al., Nanodelivery of nucleic acids, Nat. Rev. Methods Primers 2, 24, (2022). Therefore, in some embodiments of this disclosure, the compositions of this disclosure are formulated as liposomes. Liposomes are enclosed nanoparticles with a bilayer membrane that have demonstrated excellent performance in delivering nucleic acid drugs (such as DNA and RNA, such as srRNA) and various drug types. More information on this topic can be found, for example, in Hsairat H. et al., OpenNano, Vol. 11, May 2023 and Gao Y. et al., Pharmaceutics, 15(1):178, Jan 2023. In addition to liposomes, other adjuvants known in the art may be used. Those skilled in the art will understand that adjuvants can prevent rapid antigen diffusion by isolating antigens (such as nucleic acid constructs, vectors, srRNA molecules) in a localized precipitate, or they may contain substances that stimulate host secretion of factors that have chemotactic effects on macrophages and other components of the immune system.

[0157] The compositions disclosed herein can be formulated into forms compatible with their intended route of administration, such as exosomes, liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, polymers, viral replicon particles (VRPs), microspheres, immunostimulatory complexes (ICOMs), bioactive ligand conjugates, or any combination thereof. For example, in some embodiments, the compositions of this disclosure are formulated as liposomes. In some embodiments, the compositions of this disclosure are formulated as lipid-based nanoparticles (LNPs). In some embodiments, the compositions of this disclosure are formulated as polymer nanoparticles. In some embodiments, the compositions are immunogenic compositions, for example, compositions that can stimulate an immune response against EBV in a subject. In some embodiments, the pharmaceutical compound is formulated as an adjuvant. In some embodiments, the immunogenic composition is formulated as a vaccine. As used herein, the term "vaccine" refers to prophylactic or therapeutic immunization against EBV in an individual. Vaccines according to this disclosure immunize an individual against EBV infection and EBV-related diseases. Immunization involves the stimulation and sensitization of the immune system to antigens within the vaccine. According to this disclosure, prophylactic immunization refers to the initial exposure of an individual's immune system (such as a nascent immune system) to an EBV antigen. This initial exposure results in the clearance of the antigen from the exposed individual's body and the development of EBV antigen-specific CD4+- and CD8+- cells, as well as antibody-producing memory B cells. Upon a second exposure, the immune system is able to more effectively prevent and / or clear EBV infection, thereby preventing or mitigating the development of EBV-related diseases. Specifically, the effects of prophylactic immunization manifest in at least one aspect: preventing immunized individuals from contracting EBV, modifying or limiting infection, aiding, improving, enhancing, or stimulating the individual's recovery from infection, and generating immune memory that will prevent or limit subsequent EBV infection. The presence of any of these effects can be tested and detected using conventional methods known to those skilled in the art. For example, an individual is challenged using one or more EBV antigens already used as part of a vaccine, and antibody titers and T cell counts against said one or more antigens are determined. Furthermore, the in vitro induction of neutralizing antibodies that inhibit human B cell infection can be determined.

[0158] While both evoke an immune response against EBV antigens, therapeutic immunization according to this disclosure can be administered to individuals exposed to EBV prior to said immunization, for example, those already infected with EBV. In this case, the immunization leads to the reactivation of resting T effector cells that confront homologous antigens in the form of specialized antigen-presenting cells that present these antigens as MHC class I and / or MHC class molecules. Therapeutic immunization against EBV can prove particularly valuable without requiring viral reactivation, for example in patients undergoing transplantation or otherwise immunocompromised (e.g., HIV-positive individuals, cancer patients, patients with severe inflammation or autoimmune diseases), or where EBV reactivation can cause or has caused the development of diseases such as non-Hodgkin's lymphoma and post-transplant lymphoproliferative disorder (PTLD), oral hairy leukoplakia, chronic active EBV infection (CAEBV), or the B-cell transforming capacity of EBV to cause the development of diseases such as cancer.

[0159] In some embodiments, the methods of this disclosure include administration of an srRNA-based pharmaceutical composition as described herein for EBV infection, which elicits potent neutralizing antibodies and robust T-cell responses against EBV antigens, inhibits the production of viral immunomodulatory factors, and / or prevents viral latency.

[0160] In some embodiments, the srRNA-based pharmaceutical compositions (such as vaccines) disclosed herein are administered therapeutically, such as after EBV infection (to treat said infection). In some embodiments, the vaccines disclosed herein may be administered prophylactically to prevent or reduce the frequency of Hodgkin's lymphoma, Burkitt's lymphoma, gastric cancer, nasopharyngeal carcinoma, post-transplant lymphoproliferative disorders, diffuse B-cell lymphoma, and / or NK / T-cell lymphoma.

[0161] As described in more detail below, in some embodiments of this disclosure, the compositions disclosed herein can be formulated with a delivery medium into a delivery system, wherein the delivery system may comprise one or more of the following: physiological buffer, liposomes, viral replicon particles (VRP), lipid-based nanoparticles (LNP), polymer nanoparticles, physiological buffer, microspheres, immunostimulatory complexes (ISCOM), conjugates of bioactive ligands, or any combination thereof.

[0162] In some embodiments, the nucleic acid constructs of this disclosure can be delivered to cells or subjects via lipid-based nanoparticles (LNPs). LNPs are generally less immunogenic than viral particles. While many people have pre-existing immunity to viral particles, they do not have pre-existing immunity to LNPs. Furthermore, adaptive immune responses to LNPs are unlikely to occur, allowing for repeated administration of LNPs.

[0163] The lipids suitable for use in the compositions and methods described herein can be cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

[0164] In some embodiments, the LNP of this disclosure may comprise one or more ionizable lipids. As used herein, the term "ionizable lipid" refers to a lipid that is cationic when the pH drops below the pKa of its ionizable group, or becomes ionizable (protonated) but more neutral at higher pH values. At pH values ​​below the pKa, the lipid is capable of associating with negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes lipids that exhibit a positive charge when the pH decreases from physiological pH, and any of a variety of lipids that carry a net positive charge at selective pH (e.g., physiological pH). Permanent cationic lipids such as DOTMA have been shown to be too toxic for clinical use. According to other embodiments, ionizable lipids may be present in the lipid formulation, preferably at a ratio of about 30 to about 70 mol%, in some embodiments about 30 mol%, in others about 40 mol%, in still others about 45 mol%, in others about 47.5 mol%, in others about 50 mol%, in yet another embodiment, and in yet another embodiment about 60 mol% (“Mol%” means the percentage of a particular component in the total moles). The term “about” in this paragraph means a positive or negative range of 5 mol%. DODMA or 1,2-dioleoyloxy-3-dimethylaminopropane is an ionizable lipid, and DLin-MC3-DMA or O-(Z,Z,Z,Z-heptadecane-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) (“MC3”) is also an ionizable lipid.

[0165] In some embodiments, the LNP of this disclosure may comprise one or more ionizable cationic lipids. Several ionizable cationic lipids suitable for manufacturing LNPs have been developed. These cationic lipids include C12-200, MC3, LN16, and MD1, among others. Any of these cationic lipids can be used to formulate LNPs for delivery of the nucleic acid constructs of this disclosure. For example, in one type of LNP of this disclosure, the GalNAc moiety may be attached to the exterior of the LNP and act as a ligand for uptake into the liver via the desialylate glycoprotein receptor.

[0166] In some implementations, LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids (such as membrane-fused phospholipids (DOPE) or membrane component cholesterol) can be included in the LNP as "helper lipids" to enhance transfection activity and nanoparticle stability. This is because, in some cases, limitations of cationic lipids include low efficacy due to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses. LNPs can also contain hydrophobic lipids, hydrophilic lipids, or both.

[0167] In some embodiments, lipids or combinations of lipids known in the art suitable for producing LNP include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Non-limiting examples of suitable cationic lipids include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Non-limiting examples of suitable neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Non-limiting examples of suitable PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0168] In some embodiments, lipids can be combined in any molar ratio to produce the LNPs of this disclosure. Additionally, one or more polynucleotides can be combined with one or more lipids in a wide range of molar ratios to produce the LNPs of this disclosure. For example, in some embodiments, the lipid-to-RNA mass ratio of the LNP delivery system is from about 100:1 to about 4:1. In some embodiments, the delivery system comprises lipid-based nanoparticles with an average diameter of less than 1000 nm, less than 500 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 75 nm, less than 50 nm, or less than 25 nm. In some embodiments, the LNP delivery system comprises lipid-based nanoparticles with an average diameter of about 1-1000 nm, about 1-1000 nm, about 1-500 nm, about 1-250 nm, about 25-200 nm, about 25-100 nm, about 35-75 nm, or about 25-60 nm.

[0169] In some embodiments, the pharmaceutical composition is formulated for one or more of the following routes of administration: intranasal, transdermal, intrathecal, intraperitoneal, intramuscular, intratracheal, intranodular, intratumoral, intra-articular, intravenous, subcutaneous, intravaginal, intraocular, rectal, and oral. In some specific embodiments of this disclosure, the composition is formulated for intramuscular administration.

[0170] Suitable pharmaceutical compositions for injection comprise sterile aqueous solutions or dispersions, and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsipani, New Jersey), or phosphate-buffered saline (PBS). In these cases, the composition should be sterile and fluid in a manner that facilitates syringeability and / or injection. It should be stable under manufacturing and storage conditions and resistant to contamination by microorganisms such as bacteria and fungi. In the compositions disclosed herein, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, appropriate flowability can be maintained by using coatings (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants (e.g., sodium dodecyl sulfate). Microbial protection can be achieved through various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), and / or sodium chloride, are typically included in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0171] Sterile injectable solutions can be prepared by incorporating the active compound, as needed, into a suitable solvent having one or a combination of the components listed above, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and other desired components from those listed above.

[0172] In some embodiments, the pharmaceutical compositions disclosed herein, such as vaccine compositions, are formulated for inhalation, such as aerosols, sprays, mists, liquids, or powders. Administration by inhalation may be in the form of a dry powder or aerosol formulation, which is inhaled by a subject (e.g., a patient) using an inhalation device (e.g., a micro-sprayer, a pressurized metered inhaler, or a nebulizer).

[0173] In some embodiments, the composition is formulated for one or more of the following routes of administration: intranasal, intrathecal, transdermal, intramuscular, intranodal, intravenous, intraperitoneal, oral, intravaginal, and intracranial. In some embodiments, the administered composition results in an increase in interferon production in the subject. In some embodiments, the administered composition induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, the one or more pro-inflammatory molecules comprise interferon α (IFNa), interferon γ (IFN), TNF-α, IL-12, granzyme B, perforin, or any combination thereof. The method disclosed herein

[0174] Administration of any of the therapeutic compositions described herein (such as nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions) can be used to treat health conditions related to EBV infection, such as proliferative disorders (e.g., cancer), infectious diseases (e.g., infectious mononucleosis, acute or chronic infections), and / or autoimmune and / or inflammatory diseases. In some embodiments, nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as described herein can be effectively used to modulate, for example, induce or inhibit pharmacodynamic effects in a subject. Non-limiting examples of pharmacodynamic effects include immunogenic effects, biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesired effects, adverse effects, and effects in a disease or disease model. Therefore, one aspect of this disclosure relates to a method of modulating pharmacodynamic effects in a subject with such need, the method comprising administering to the subject a composition comprising one or more of the following: (a) a nucleic acid construct as described herein (e.g., a replicon, such as a self-replicating RNA construct); (b) a recombinant cell as described herein; and (c) a pharmaceutical composition as described herein. In some embodiments, the pharmacodynamic effect includes one or more of the following: immunogenic effect, biomarker response, therapeutic effect, prophylactic effect, desired effect, undesired effect, adverse effect, and effect in a disease model. In some embodiments, the pharmacodynamic effect includes prevention of viral infection. In some embodiments, the pharmacodynamic effect includes reducing the viral load of a subject. In some embodiments, the pharmacodynamic effect includes inducing an immune response in a subject. Those skilled in the art will understand that multiple aspects of an immune response can be induced in the disclosed methods. For example, in some embodiments, the induced immune response includes an anti-EBV antibody response, a neutralizing antibody response, and an anti-EBV T-cell response, or any combination thereof.

[0175] In some embodiments, nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as described herein may be incorporated into therapeutic agents for use in methods of treating subjects who have, are suspected of having, EBV-related health conditions or diseases (such as inflammatory diseases, autoimmune diseases, or cancer), or who are at high risk of developing EBV-related health conditions or diseases (such as inflammatory diseases, autoimmune diseases, or cancer). Therefore, in another aspect, this document provides methods for preventing or treating health conditions in subjects, the methods comprising prophylactically or therapeutically administering a composition to the subject comprising one or more of the following: (a) a nucleic acid construct (e.g., a replicon, such as a self-replicating RNA construct) as described herein; (b) a recombinant cell as described herein; and (c) a pharmaceutical composition as described herein. In some embodiments, the administered composition elicits an immune response in the subject. In some embodiments, the administered composition induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, the one or more pro-inflammatory molecules include interferon α (IFNa), interferon γ (IFN), TNF-α, IL-12, granzyme B, perforin, or any combination thereof. In some embodiments, the administered composition confers (e.g., causes) an increase in interferon production in the subject. In some embodiments, the subject has or is suspected of having EBV-related disease. In some embodiments, the subject has previously received one or more therapies and has developed at least partial resistance to said one or more therapies.

[0176] Exemplary health conditions or diseases may include, but are not limited to, cancer, rare diseases, immune diseases, autoimmune diseases, acute and chronic infections, and inflammatory diseases such as infectious mononucleosis (IM), characterized by the abnormal proliferation of EBV-infected B cells, which leads to the massive activation and proliferation of immune cells, resulting in lymphoid hyperplasia, tonsillitis, lymphadenopathy, and hepatosplenomegaly. In some embodiments, the subject is a patient under the care of a physician.

[0177] Examples of autoimmune and inflammatory diseases suitable for the methods of this disclosure include, but are not limited to, rheumatoid arthritis, osteoarthritis, Still's disease, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, systemic lupus erythematosus, Sjogren's syndrome, diabetic retinopathy, diabetic vasculopathy, diabetic neuralgia, insulitis, psoriasis, alopecia areata, warm and cold autoimmune hemolytic anemia (AIHA), pernicious anemia, and acute inflammatory diseases. Inflammatory diseases, autoimmune adrenalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, lichen sclerosis, Graves' disease, Behçet's disease, Ménière's disease, reactive arthritis (Rett syndrome),Reiter's syndrome, Chug-Strauss syndrome, Cogan syndrome, CREST syndrome, pemphigus vulgaris and pemphigusfoliaceus, bullous pemphigoid, polymyalgia, polymyositis, primary biliary cirrhosis, pancreatitis, peritonitis, psoriatic arthritis, rheumatic fever, sarcoidosis, Sjörgensen syndrome, scleroderma, celiac disease, stiff-man syndrome, Takayasu arteritis. Arteritis, transient gluten intolerance, autoimmune uveitis, vitiligo, polychondritis, dermatitis herpetiformis (DH) or Duhring's disease, fibromyalgia, Goodpasture syndrome, Guillain-Barré syndrome, Hashimoto's thyroiditis, autoimmune hepatitis, inflammatory bowel disease,IBD, Crohn's disease, ulcerative colitis, myasthenia gravis, immune complex disorders, glomerulonephritis, polyarteritis nodosa, antiphospholipid syndrome, polyglandular autoimmune syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), urticaria, autoimmune infertility, juvenile rheumatoid arthritis, sarcoidosis, and autoimmune cardiomyopathy.

[0178] Non-limiting examples of inflammatory diseases for which the methods of this disclosure may be applied to treatment or prevention include inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, rheumatoid arthritis, polymyositis, and myocarditis. In some embodiments, the inflammatory disease is infectious mononucleosis (IM), characterized by the abnormal proliferation of EBV-infected B cells, which leads to massive activation and proliferation of immune cells, resulting in lymphoid hyperplasia, tonsillitis, lymphadenopathy, and hepatosplenomegaly.

[0179] In some embodiments, the health condition is a proliferative disorder or a viral infection (such as EBV infection). In some embodiments, the subject has or is suspected of having a condition associated with a proliferative disorder or viral infection. In some embodiments, the health condition is a proliferative disorder, which is a lymphoproliferative disorder or cancer associated with EBV infection, such as post-transplant lymphoproliferative disorder (PTLD), Hodgkin's lymphoma, Burkitt's lymphoma, and nasopharyngeal carcinoma.

[0180] In some embodiments, the health condition is a rare disease (e.g., a disease or condition affecting fewer than 200,000 people in the United States, as defined by The Orphan Drug Act (www.fda.gov / patients / rare-diseases-fda)) and / or an inflammatory disease and / or an autoimmune disease. In some embodiments, the subject has or is suspected of having a condition associated with an inflammatory disease and / or an autoimmune disease and / or a rare disease (such as, but not limited to, chronic active Epstein-Barr virus (CAEBV) disease).

[0181] In some embodiments, the compositions of this disclosure are formulated to be compatible with their intended route of administration. For example, the nucleic acid constructs (such as srRNA constructs), recombinant cells, and / or pharmaceutical compositions of this disclosure may be administered orally or by inhalation, but are more likely to be administered via a parenteral route. Examples of parenteral administration routes include, for example, intramuscular, intratumoral, intravenous, intranodular, intradermal, subcutaneous, transdermal (local), transmucosal, intravaginal, and rectal administration. In some embodiments, the compositions are administered intramuscularly. In some embodiments, the compositions are administered intratumorally. Solutions or suspensions intended for parenteral administration may contain the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, phosphates, tris, sucrose, and agents for tonication (such as sodium chloride or dextrose). The pH may be adjusted (e.g., to approximately 7.2-7.8, such as 7.5) with acids or bases (e.g., sodium monohydrogen phosphate and / or sodium dihydrogen phosphate, hydrochloric acid, or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0182] The dosage, toxicity, and therapeutic efficacy of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of this disclosure can be determined using standard pharmaceutical procedures in cell culture or laboratory animals, for example, to determine the LD50 (the dose that is lethal to 50% of the population) and ED. 50 (The dose effective for 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, and it can be expressed as the ratio LD50. 50 / ED 50 This indicates that compounds exhibiting a high therapeutic index are generally suitable. While compounds exhibiting toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the affected tissue site to minimize potential damage to uninfected cells, thereby reducing side effects.

[0183] For example, data obtained from cell culture experiments and animal studies can be used to formulate a range of dosages for human use. Dosages of such compounds are typically within the range of circulating concentrations, including those with low or no toxicity, such as endocrine disruptors (EDS). 50 The dosage may vary within this range depending on the dosage form and route of administration used. For any compound used in the methods of this disclosure, the therapeutically effective dose can initially be estimated by cell culture measurements. Doses can be formulated according to animal models to achieve a range of circulating plasma concentrations, which includes the IC50 values ​​determined in cell culture. 50 (For example, the concentration of a test compound that achieves half-maximal inhibition of symptoms). This type of information can be used to more accurately determine the effective dose in the human body. Levels in plasma can be measured, for example, by high-performance liquid chromatography.

[0184] The therapeutic compositions described herein (such as nucleic acid constructs (e.g., srRNA constructs), recombinant cells, and / or pharmaceutical compositions) may be administered once or multiple times. Those skilled in the art will understand that certain factors may affect the dosage and duration required for effective treatment of a subject, including but not limited to the severity of the disease, prior treatment, the subject's overall health and / or age, and any other pre-existing conditions. Additionally, treatment of a subject with a therapeutically effective amount of the pharmaceutical composition of this disclosure may comprise monotherapy or a series of treatments. In some embodiments, the composition is administered once or multiple times, followed by a rest period of 2 to 56 days (e.g., 28 days), followed by additional administration every 2 to 12 months. Repeat administration may be performed every 1 to 10 years. Regarding nucleic acid constructs (e.g., srRNA constructs), the therapeutically effective amount (e.g., effective dose) of the nucleic acid construct of this disclosure depends on the selected nucleic acid construct. For example, a single dose ranging from about 0.001 to 0.1 mg / kg of the patient's body weight may be administered. In some embodiments, about 0.005, 0.01, or 0.05 mg / kg may be administered. In some embodiments, a single dose ranging from about 0.001 μg to 300 μg / kg of the patient's body weight may be administered. In some embodiments, a single dose ranging from about 0.3 mg to 3 mg / kg of the patient's body weight may be administered.

[0185] As discussed above, a therapeutically effective amount includes an amount sufficient to promote a specific effect when the therapeutic composition is administered to a subject (e.g., a subject who has, is suspected of having, or is at risk of developing a health condition such as cancer or infection). In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of symptoms of a disease or infection, alter the course of symptoms of a disease or infection (e.g., but not limited to, slowing the progression of symptoms of a disease or infection), or reverse the symptoms of a disease or infection. It should be understood that, for any given situation, a person skilled in the art can determine an appropriate effective amount using conventional experiments.

[0186] The therapeutic efficacy of therapeutic compositions including those disclosed herein for treating diseases or infections can be determined by a qualified clinician. However, treatment may be considered effective if at least one or all signs or symptoms of the disease or infection improve or are alleviated. Efficacy can also be measured by failure of individual deterioration as assessed by hospitalization or the need for medical intervention (e.g., cessation or at least slowing of disease or infection progression). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of a disease or infection in a subject or animal (some non-limiting examples include humans or mammals) and includes: (1) suppressing the disease or infection, e.g., stopping or slowing the progression of symptoms; or (2) alleviating the disease or infection, e.g., causing the resolution of symptoms; and (3) preventing or reducing the likelihood of symptom development.

[0187] In some embodiments, the nucleic acid constructs (such as srRNA constructs), recombinant cells, and / or pharmaceutical compositions of this disclosure may be administered to a subject in a pharmaceutically acceptable carrier and in an effective amount to stimulate an immune response. Typically, the subject may be immunized via an initial series of injections (or administered via one of the other routes described below), followed by a booster to enhance the protection provided by the initial series of administrations. Such dosage and timing of the initial series of injections and the subsequent booster are necessary to stimulate an immune response in the subject. In some embodiments, the administered composition results in an increase in the subject's interferon production. In some embodiments of the disclosed methods, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0188] As described above, pharmaceutically acceptable carriers suitable for injectable applications include sterile aqueous solutions (when water-soluble) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In these cases, the composition must be sterile and must be a fluid to a degree of ease of perfusion. The composition must further be stable under manufacturing and storage conditions and must resist contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof, and vegetable oils. For example, appropriate flowability can be maintained by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Antimicrobial activity can be achieved by various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.).

[0189] Sterile injectable solutions can be prepared by incorporating nucleic acid constructs, recombinant cells and / or recombinant peptides in the required amounts into a suitable solvent having one or a combination of the components listed above, followed by filtration and sterilization.

[0190] As described above, nucleic acid constructs, recombinant cells, recombinant peptides, and / or pharmaceutical compositions, when properly protected, can be administered orally, for example, with an inert diluent or an absorbable, edible carrier. Nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions, along with other ingredients, can also be encapsulated in hard or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into an individual's diet. For oral therapeutic administration, the active compound can be combined with excipients and used in the form of ingestible tablets, sublingual tablets, lozenges, capsules, elixirs, suspensions, syrups, cake formulations, etc.

[0191] As described above, in some embodiments of this disclosure, the compositions disclosed herein (such as the nucleic acid constructs of this disclosure) can be formulated together with a delivery medium to form a delivery system, wherein the delivery system comprises one or more of the following: physiological buffer, liposomes, viral replicon particles (VRP), lipid-based nanoparticles (LNP), polymer nanoparticles, physiological buffer, microspheres, immunostimulatory complexes (ISCOM), conjugates of bioactive ligands, or any combination thereof.

[0192] In some embodiments, the nucleic acid constructs of this disclosure can be delivered to cells or subjects via lipid-based nanoparticles (LNPs). LNPs are generally less immunogenic than viral particles. While many people have pre-existing immunity to viral particles, they do not have pre-existing immunity to LNPs. Furthermore, adaptive immune responses against LNPs are unlikely to occur, allowing for repeated administration of LNPs.

[0193] In some embodiments, the therapeutic compositions described herein (such as nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions) are incorporated into therapeutic compositions for use in methods of preventing or treating subjects who have, are suspected of having, cancer, autoimmune diseases, inflammatory diseases, and / or infections, or who are at high risk of developing cancer, autoimmune diseases, inflammatory diseases, and / or infections. In some embodiments, the microbial infection is a viral infection, such as EBV infection. Other treatments

[0194] In some embodiments, the composition according to this disclosure is administered to the subject alone as a monotherapy (single therapy) or in combination with at least one other therapy (e.g., a second therapy) as a first therapy. In some embodiments, the second therapy is selected from the group consisting of: chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from the group consisting of: chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first therapy and the second therapy are administered concurrently. In some embodiments, the first therapy and the second therapy are administered at the same time. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered alternately. In some embodiments, the first therapy and the second therapy are administered together in a single formulation. Reagent test kit

[0195] This document also provides various kits for practicing the methods described herein, as well as written instructions for manufacturing and using the kits. In particular, some embodiments of this disclosure provide kits for inducing pharmacodynamic effects. Some embodiments of this disclosure provide kits for inducing an immune response in a subject. Some other embodiments relate to kits for preventing health conditions in subjects with this need. Some other embodiments relate to kits for methods of treating cancer in subjects with this need. For example, in some embodiments, this disclosure provides kits comprising one or more of nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as provided and described herein, along with written instructions for preparing and using the kit. In some embodiments, the kits are used for the prevention and / or treatment of one or more EBV-related diseases as described above. In some specific embodiments, the EBV-related diseases include infectious mononucleosis (IM), autoimmune and / or inflammatory diseases and / or rare diseases and / or cancer.

[0196] In some embodiments, the kit of this disclosure further includes one or more means for administering any one of the provided nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kit of this disclosure further includes one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) for administering any one of the provided nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions to a subject. In some embodiments, the kit may have one or more additional therapeutic agents that may be administered simultaneously or sequentially with other kit components for a desired purpose, such as inducing an immune response, preventing and / or treating a health condition in a subject with this need.

[0197] Any of the above kits may further include one or more additional reagents, wherein such additional reagents may be selected from: dilution buffers; reconstitution solutions, washing buffers, control reagents, control expression vectors, negative controls, positive controls, and reagents suitable for the in vitro production and / or administration of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the present disclosure.

[0198] In some embodiments, the kit components may be in separate containers. In some other embodiments, the kit components may be combined in a single container. Thus, in some embodiments of this disclosure, the kit comprises one or more of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions provided and described herein in one container (e.g., in a sterile glass or plastic vial) and additional therapeutic agents in another container (e.g., in a sterile glass or plastic vial).

[0199] In another embodiment, the kit comprises a combination of compositions described herein, including one or more nucleic acid constructs, recombinant cells and / or recombinant peptides of this disclosure, and one or more additional therapeutic agents, optionally in the form of a pharmaceutical formulation in a single common container.

[0200] In some embodiments, the kit includes a pharmaceutical composition for parenteral administration to a subject, and the kit may include a device (such as an injection device or catheter) for such administration. For example, the kit may include a subcutaneous injection needle or other injection device as discussed above, containing one or more nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of this disclosure.

[0201] In some embodiments, the kit may also include instructions for using the kit components to practice the methods disclosed herein. For example, the kit may include a packaging insert containing information about the pharmaceutical compositions and dosage forms in the kit. Typically, such information helps patients and physicians use the packaged pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding combinations of this disclosure may be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, how to supply, appropriate storage conditions, references, manufacturer / distributor information, and intellectual property information.

[0202] Instructions for practicing the methods are typically documented on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may exist as a packaging insert in the kit, on the container of the kit, or on the label of its components (e.g., associated with packaging or repackaging). The instructions may exist as an electronic storage data file on a suitable computer-readable storage medium (e.g., CD-ROM, floppy disk, flash drive, etc.). In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the Internet) are provided. An example of this embodiment is a kit that includes a URL where the instructions can be viewed and / or downloaded. Like the instructions themselves, such means for obtaining the instructions may be documented on a suitable substrate or recording medium.

[0203] All publications and patent applications mentioned in this disclosure are incorporated herein by reference as if each individual publication or patent application were specifically and individually incorporated by reference.

[0204] No references cited herein are acknowledged to constitute prior art. The discussion of these references presents the claims made by their authors, and the applicant reserves the right to challenge the accuracy and relevance of the cited documents. It should be clearly understood that although numerous sources of information are mentioned herein, including scientific journal articles, patent documents, and textbooks, such mentions do not imply an admission that any of these documents constitutes part of common general knowledge in the art.

[0205] The discussion of the general methods presented herein is for illustrative purposes only. Other alternative methods and solutions will be apparent to those skilled in the art upon review of this disclosure and will be included within the spirit and scope of this application.

[0206] Further embodiments are disclosed in detail in the following examples, which are provided by way of illustration only and are not intended to limit the scope of this disclosure or the claims in any way. Example

[0207] Unless otherwise stated, the practice of this disclosure will employ conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology that are well known to those skilled in the art. These techniques are explained in detail in the literature, such as Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, DM et al., (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al., (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, MG et al., (1995). Viral Vectors: GeneTherapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The ComprehensiveSourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al., (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology.New York, NY: Wiley; Mullis, KB, Ferré, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley (including supplements through 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the contents of which are incorporated herein by reference.

[0208] Further embodiments are disclosed in detail in the following examples, which are provided by way of illustration only and are not intended to limit the scope of this disclosure or the claims in any way. Example 1 Construction of modified alphavirus vector

[0209] This embodiment describes experiments conducted to construct a basic alphavirus vector (e.g., without a heterologous gene), which is then used to construct an alphavirus vector encoding a polypeptide construct of interest, such as a polypeptide construct containing one or more antigenic determinants derived from Epstein-Barr virus (EBV).

[0210] The basic VEEV vector was synthesized de novo from a reference sequence (Genbank L01443) of the TC-83 strain carrying a silent A2087G mutation, consisting of four approximately 4 kb portions (Twist Bioscience, Thermo Fisher GeneArt). The coding sequence of the VEEV structural gene was replaced with a unique SpeI restriction site (where 5'A is the nucleotide following the P2A sequence after nucleotide 93 of the structural polyprotein gene, and 3'T matches the position of the TGA stop codon of the structural polyprotein). A 5' adaptor sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 59) was inserted upstream of the SpeI site, and a 3' adaptor sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 60) was inserted downstream of the SpeI site for subsequent Gibson Assembly® procedures. The phage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 61) is contained upstream of the VEEV genome sequence, and downstream of it is a poly(A) sequence, followed by a SapI site upstream of its cleavage recognition site. Immediately downstream of the SapI site is the T7 terminator sequence (5'-AACCCCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 62), followed by a unique NotI restriction enzyme site. These parts are assembled in five Gibson Assembly® reactions (e.g., a linearized pYL backbone and four synthetic fragments) to form the VEEV basic vector.

[0211] The EBV gene was codon-optimized for human expression on an intracytoplasmic transgenetic de novo (IDT) basis. The synthesized product was amplified using primers with 5′ and 3′ adaptor sequences added to the gene ends, or primers with sequences homologous to adjacent gene inserts. The SpeI linearized base vector and PCR product were combined using the Gibson Assembly® program to form the final vector. Figure 2The diagram shows twenty-two (22) exemplary nucleic acid constructs that express EBV antigenic determinants derived from one or more EBV polypeptide genes. In each construct, the number of EBV antigens and their order of arrangement in the antigen cassette are shown. Some constructs contain coding sequences for multiple polypeptides that are operatively interconnected within a single polycistronic ORF (see, for example, Rep-674, Rep-675, Rep-678, Rep-679, Rep-680, Rep 728, Rep-729, Rep-730, and Rep-731). Some other constructs comprise coding sequences for multiple peptides configured in two ORFs interconnected by sequences from the internal ribosome entry site (IRES) of the ECMV (see, for example, Rep-692, Rep-693, Rep-694, Rep-696, Rep-697, Rep-698, Rep-699, Rep-733, Rep-734, Rep-735, Rep-736, and Rep-737). Figure 2 In the image, the EBV polypeptide, the P2A sequence of the autologous protein hydrolysate, and the internal ribosome entry site (IRES) are displayed in the direction from the N-terminus to the C-terminus of the corresponding coding sequence (i.e., the 5' to 3' direction). Example 2 In vitro evaluation of srRNA constructs expressing selected EBV antigens

[0212] This embodiment describes in vitro experiments conducted to evaluate the expression level and biological activity of the synthetic srRNA construct described in Example 1 above, and to investigate any differential behaviors (such as replication and protein expression).

[0213] In vitro transcription: In vitro transcription of the SapI linearized plasmid template was performed using phage T7 RNA polymerase (HiScribe™ T7 High-Yield RNA Synthesis Kit, NEB), followed by the addition of 5' cap 1 (vaccinia capping system, mRNA cap 2'-O-methyltransferase, NEB) to prepare RNA. RNA was purified using LiCl precipitation, followed by washing with 70% ethanol and resuspending in 1 mM sodium citrate at pH 6.4. RNA concentration was determined by absorbance at 260 nm (spectrophotometer, Thermo Fisher Scientific).

[0214] Biological activity (RNA replication):srRNA-based bioactivity was measured by detecting replication intermediate dsRNA in BHK-21 cells. RNA was transformed into cells via electroporation (4D-Nucleofector™, Lonza). Cells were fixed and permeabilized (eBioscience™ Foxp3 / transcription factor staining buffer kit, Invitrogen) 15–22 hours post-transformation and stained with a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Schicons). The frequency of dsRNA+ cells was quantified by fluorescence flow cytometry. Figure 3 The frequency of dsRNA+ cells was plotted for each construct, indicating that the EBV srRNA vaccine construct exhibits RNA replication.

[0215] Protein expression (gp350 and gH / gL): In these experiments, protein expression was detected by flow cytometry using antibodies that recognize the gp350 and gH / gL complexes. The mean fluorescence intensity (MFI) of the fluorescently labeled antibodies was plotted as a measure of protein expression. RNA was transformed into BHK-21 cells via electroporation (e.g., 4D-Nucleofector™, Lonza). 15–22 hours post-transformation, cells were fixed and permeabilized (eBioscience™ Foxp3 / transcription factor staining buffer kit, Invitrogen) and stained with IgG2a anti-gp350 antibody, followed by staining with APC-conjugated anti-IgG2a secondary antibody, and then with PE-Cy7-conjugated anti-gH / gL antibody. The MFI of gp350+ and gH / gL+ cells was measured by flow cytometry, and the results were recorded in [data missing]. Figure 4 The figure shows the relative expression of three of the five antigens in various multi-gene or single-gene srRNA vaccine constructs in electroporated BHK-21 cells.

[0216] Protein expression (BZLF1): In these experiments, protein expression was detected by Western blotting of whole-cell lysates from electroporated BHK-21 cells using an antibody against BZLF1. RNA was transformed into BHK-21 cells via electroporation (e.g., 4D-Nucleofector™, Lonza), and RNA was collected for lysis 15–22 hours post-transformation. Protein content in the lysates was measured using the bisquinolinic acid assay (BCA), normalized, and then run on electrophoresis gels in duplicate. After transfer to nitrocellulose membranes, the membranes were excised and detected using probes with either an anti-BZLF1 antibody or an anti-actin antibody. Results of various single-gene and multi-gene srRNA vaccine constructs from electroporated BHK-21 cells were presented in […]. Figure 5 It is displayed in the middle. Figure 5The relative expression of BZLF1 in different constructs is shown (left), with observed actin staining (right) as a control.

[0217] These data indicate that the EBV srRNA vaccine construct underwent RNA replication ( Figure 3 ), and express gp350, gH / gL (gp350, gH / gL), which are required to trigger an immune response. Figure 4 ) and BZLF1 ( Figure 5 )antigen. Example 3 srRNA constructs expressing selected EBV antigens elicit antibody responses in vivo.

[0218] This embodiment describes an experiment conducted to demonstrate that an srRNA-based vaccine can elicit an antibody response in vivo to the encoded EBV antigen.

[0219] LNP formulation srRNA was formulated into lipid nanoparticles using a microfluidic mixer, and particle size and polydispersity were analyzed using dynamic light scattering, with encapsulation efficiency determined by RiboGreen® (dye exclusion assay). Lipids were suspended in ethanol. RNA was suspended at a concentration of 103 μg / mL in 50 mM citrate at pH 4 and mixed at a 3:1 flow ratio (water:organic matter) at a total flow rate of 12 mL / min.

[0220] Mice and injection CD1 ISG mice were purchased from Charles River Labs, Envigo, or Jackson Laboratories. On the day of administration, 1 μg of material was injected intramuscularly into one quadriceps femoris muscle or divided into two portions and injected into both quadriceps femoris muscles. Animals were administered the material on days 0 and 28. Throughout the study, animal weight and other general observations were monitored.

[0221] Antibody reaction. In vivo antibody responses to the EBV surface antigen gH / gL complex and gp350 encoded on various srRNA vaccine constructs were quantified using an IgG ELISA. In these experiments, serum from immunized mice was collected 41 days after the first administration for measurement. Responses were quantified using standard mouse antibodies against each EBV antigen. Figure 6A The results of the anti-gH / gL IgG response were shown, and Figure 6B Results of the anti-gp350 IgG response are shown, a direct ELISA method using serum from immunized mice against EBV protein gp350 and the gH / gL complex.

[0222] These data indicate that srRNA-based vaccines can generate an immune response in vivo in the form of antibody responses against the EBV surface antigen gH / gL complex and gp350. Example 4 srRNA constructs expressing selected EBV antigens elicit T cell responses in vivo.

[0223] This embodiment describes an experiment conducted to demonstrate that srRNA-based vaccines can generate T-cell responses against encoded EBV antigens in vivo.

[0224] LNP formulation. srRNA was formulated into lipid nanoparticles using a microfluidic mixer, and particle size and polydispersity were analyzed using dynamic light scattering (DLS), while encapsulation efficiency was determined using RiboGreen® (dye exclusion assay). Lipids were suspended in ethanol. RNA was suspended at a concentration of 103 μg / mL in 50 mM citrate at pH 4 and mixed at a 3:1 flow ratio (water:organic matter) at a total flow rate of 12 mL / min.

[0225] Mice and injection. CD1 ISG mice were purchased from Charles River Labs, Envigo, or Jackson Laboratories. On the day of administration, 1 μg of material was injected intramuscularly into one quadriceps femoris muscle or divided into two portions and injected into both quadriceps femoris muscles. Animals were administered the material on days 0 and 28. Throughout the study, animal weight and other general cage-side observations were monitored. Serum was collected throughout the study for analysis of anti-EBV antibody responses. At the end of the study (day 41), spleens were collected to assess anti-EBV T-cell responses.

[0226] ELISpot. To measure the scale of EBV antigen-specific T cell responses, spleen cells were stimulated with peptide pools corresponding to each encoded antigen, and IFNγ secretion was detected using the commercially available ELISpot kit. The gH / gL ratio of T cells to the five encoded antigens was shown at 41 days after the first administration. Figure 7A ), gp350 ( Figure 7B LMP2B Figure 7C ) and BZLF1 ( Figure 7D The response of T cells is shown on the Y-axis. The total T cell response is represented by spots formed per million cells.

[0227] These data demonstrate that srRNA-based EBV vaccines can generate T-cell responses against encoded EBV antigens in vivo. Example 5 srRNA constructs expressing selected EBV antigens elicit neutralizing antibodies in vivo.

[0228] This embodiment describes an experiment conducted to demonstrate that srRNA-based vaccines can generate neutralizing antibodies against EBV in vivo.

[0229] LNP formulations. srRNA was formulated into lipid nanoparticles using a microfluidic mixer, and particle size and polydispersity were analyzed using dynamic light scattering (DLS), while encapsulation efficiency was determined using RiboGreen® (dye exclusion assay). Lipids were suspended in ethanol. RNA was suspended at a concentration of 103 μg / mL in 50 mM citrate at pH 4 and mixed at a 3:1 flow ratio (water:organic matter) at a total flow rate of 12 mL / min.

[0230] Mice and injection. CD1 ISG mice were purchased from Charles River Laboratories, Envigo, or Jackson Laboratories. On the day of administration, the vaccine was injected intramuscularly into one quadriceps femoris muscle or divided into two portions and injected into both quadriceps femoris muscles. Animals were administered the vaccine on days 0, 56, and 120. Throughout the study, animal weight and other general cage-side observations were monitored. Serum was collected throughout the study for analysis of anti-EBV antibody responses.

[0231] EBV infection neutralization. To measure the presence of antibodies neutralizing EBV infection, serum was collected from mice that had received one, two, or three doses of srRNA vaccine and evaluated using the Raji B cell line in a virus neutralization assay. Figure 8 The 50% neutralization titer (NT50) level is shown as a bar graph representing the geometric mean, with error bars representing the geometric standard deviation. Kruskal-Wallis statistical comparisons between groups are shown.

[0232] These data indicate that srRNA-based EBV vaccines can generate a neutralizing antibody response against EBV in vivo.

[0233] While specific alternatives to this disclosure have been presented, it should be understood that various modifications and combinations are possible and can be considered within the true meaning and scope of the appended claims. Therefore, there is no intention to limit the specific abstract and disclosure presented herein.

Claims

1. A nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of said modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct, said polypeptide construct comprising one or more antigenic determinants derived from Epstein-Barr virus (EBV).

2. The nucleic acid construct according to claim 1, wherein the one or more antigenic determinants are derived from one or more EBV polypeptides / antigens, wherein the EBV polypeptides / antigens are encoded by cleavage genes, pre-latent genes, latent genes, early genes, immediate early genes, late genes, latent reactivation genes, any variants thereof, or any combination thereof.

3. The nucleic acid construct according to any one of claims 1-2, wherein the EBV latent gene is selected from the group consisting of LMP2, BKRF1, BYRF1, BLRF3 / BERF1, BERF2a / b, BERF3 / 4, BamHI-W, BNLF1 (LMP1), BARTs and EBER1 / 2.

4. The nucleic acid construct according to any one of claims 1-3, wherein the EBV latent gene is LMP2, BNLF1 (LMP1), BKRF1 (EBNA1), BLRF3 / BERF1 (EBNA3A-C, -LP) and BYRF1 (EBNA2).

5. The nucleic acid construct according to any one of claims 1-4, wherein the EBV immediate early gene is selected from the group consisting of BZLF1, BRLF1 and BMLF1.

6. The nucleic acid construct according to any one of claims 1-5, wherein the early EBV gene is selected from the group consisting of BRRF1, BORF2, BaRF1, BXLF1, BGLF5, BLLF3, BKRF3, BALF5, BMRF1, BALF2, BSLF1, BBLF2 / 3, BBLF4, BSLF2, BHRF1, BALF1, BARF1, BGLF4, BFRF1, BHLF1, BHLF2 and BNLF2a.

7. The nucleic acid construct according to any one of claims 1-6, wherein the late EBV gene is selected from the group consisting of BLLF1 (gp350 / 220), BNRF1, BPLF1, BOLF1, BVRF1, BBLF1, BGLF1, BSRF1, BRRF2, BDLF2, BKRF4, BcLF1, BDLF1, BFRF3, BLRF2, BdRF1, BBRF1, BVRF2, BGLF2, BORF1, BLRF1 (gN), BZLF2, BKRF2 (gp25, gL), BBRF3 (gM), BXLF2 (gp85, gH), BILF1, BILF2, BALF4 (gp100, gB), BDLF3, BMRF2, BALF3 and BCRF1.

8. The nucleic acid construct according to any one of claims 1-7, wherein the EBV latent gene is selected from the group consisting of BLLF1 (gp350 / 220), BXLF2 (gp85, gH), BALF4 (gp100, gB), BSLF1 (gp42) and BKRF2 (gp25, gL).

9. The nucleic acid construct according to any one of claims 1-8, wherein the pre-EBV latent gene is BYRF1 (EBNA2).

10. The nucleic acid construct according to any one of claims 1-7, wherein the one or more EBV genes are selected from the group consisting of BLLF1 (gp350 / 220), BZLF1, BXLF2 (gp85, gH), BKRF2 (gp25, gL), LMP2 and any combination thereof.

11. The nucleic acid construct according to any one of claims 1-10, wherein the coding sequence of the polypeptide construct comprises the coding sequence of the following gene: (a) BZLF1 or a variant thereof; (b) BLLF1 (gp350 / 220) or a variant thereof; (c) LMP2B or a variant thereof; (d) gH and gL, or any variant thereof; (e) BZLF1, LMP2B, gH, gL and BLLF1 (gp350 / 220), or any variant thereof; (f) gH, gL, and BLLF1 (gp350 / 220), or any variant thereof; or (g)BZLF1 and LMP2B, or any variant thereof.

12. The nucleic acid construct according to any one of claims 1-11, wherein the BLLF1 (gp350 / 220) or a variant thereof lacks a functional transmembrane (TM) domain.

13. The nucleic acid construct according to any one of claims 1-12, wherein the gH or a variant thereof lacks a functional transmembrane (TM) domain.

14. The nucleic acid construct according to any one of claims 1-13, wherein the BZLF1 or a variant thereof lacks (i) a functional nuclear localization sequence (NLS) and / or (ii) a functional transactivation (TA) domain.

15. The nucleic acid construct according to any one of claims 1-14, wherein the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34.

16. The nucleic acid construct according to any one of claims 1-15, wherein the coding sequences of the antigenic determinants are operatively linked to each other within a single open reading frame (i.e., in a polycistronic ORF).

17. The nucleic acid construct according to any one of claims 1-15, wherein the coding sequences of the antigenic determinants are operatively linked to each other within two or more ORFs.

18. The nucleic acid construct according to any one of claims 1-17, wherein the coding sequences of the antigenic determinants are operatively linked to each other via one or more adapters.

19. The nucleic acid construct according to any one of claims 18, wherein the one or more adapters comprise a sequence encoding an autologous proteolytic peptide sequence or an internal ribosome entry site (IRES).

20. The nucleic acid construct according to claim 19, wherein the autologous proteolytic peptide sequence comprises one or more autologous proteolytic cleavage sequences, wherein the autologous proteolytic sequences are derived from calcium-dependent serine endonuclease (furin), porcine cephalovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), *Melilothorax fasciatus* virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), muscular muscular dermatitis virus 2A (BmIFV2A), or combinations thereof.

21. The nucleic acid construct according to claim 19, wherein the internal ribosome entry site (IRES) is derived from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, viremia virus IRES, cricket paralysis virus IRES, gramineous aphid virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, microRNA virus IRES, encephalocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES.

22. The nucleic acid construct according to any one of claims 1-18, wherein the one or more EBV antigenic determinants are of type EBV1 (EBV-1) or type EBV2 (EBV-2) or a combination thereof.

23. The nucleic acid construct according to any one of claims 1-22, wherein the one or more EBV antigenic determinants are from a virulent EBV strain or a non-virulent EBV strain.

24. The nucleic acid construct according to any one of claims 22-23, wherein the one or more EBV antigenic determinants are derived from the AG876 strain, Akata strain, Alaska strain, B95-8 strain, Hina1 strain, HKNPC strain, China 2 strain, CVI988 strain, GD1 strain, GD2 strain, GP202 strain, M81 strain, Mediterranean strain, Mutu strain, P3HR1 strain, Raji strain, SNU-719 strain, YCCEL1 strain, or combinations thereof.

25. The nucleic acid construct according to any one of claims 1-24, wherein the modified alphavirus genome or srRNA does not contain a nucleic acid sequence encoding a viral structural protein.

26. The nucleic acid construct according to any one of claims 1-25, wherein the nucleic acid sequence encoding the polypeptide construct is operatively linked to a promoter sequence.

27. The nucleic acid construct according to claim 26, wherein the promoter sequence is a 26S subgenome (sg) promoter.

28. The nucleic acid construct according to any one of claims 1-27, wherein the srRNA is a capped srRNA containing a 5'-cap.

29. The nucleic acid construct according to claim 28, wherein the capped srRNA is a co-transcribed capped srRNA.

30. The nucleic acid construct according to claim 28, wherein the capped srRNA is an enzymatically capped srRNA.

31. The nucleic acid construct according to any one of claims 1-30, wherein the modified alphavirus genome or srRNA is an alphavirus belonging to the VEEV / EEEV group, or the SFV group, or the SINV group.

32. The nucleic acid construct according to claim 31, wherein the alphavirus is Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Chikungunya virus (CHIKV), Madariaga virus (MADV), Western equine encephalitis virus (WEEV), or Sindbis virus (SINV).

33. The nucleic acid construct according to any one of claims 1-32, wherein the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35-56.

34. A recombinant cell comprising a nucleic acid construct according to any one of claims 1-33.

35. The recombinant cell according to claim 34, wherein the recombinant cell is a eukaryotic cell.

36. The recombinant cell according to claim 34, wherein the recombinant cell is an animal cell.

37. The cell of claim 36, wherein the animal cell is a vertebrate cell or an invertebrate cell.

38. The recombinant cell according to claim 37, wherein the recombinant cell is an insect cell.

39. The recombinant cell according to claim 38, wherein the recombinant cell is a mosquito cell.

40. The recombinant cell of claim 37, wherein the recombinant cell is a mammalian cell.

41. The recombinant cell according to claim 37, wherein the recombinant cell is selected from the group consisting of: monkey kidney CV1 cells transformed with SV40, human embryonic kidney cells (HEK), young hamster kidney cells (BHK) or their derivatives, mouse Sertoli cells, monkey kidney cells, human cervical cancer cells, canine kidney cells, buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumors, TRI cells, FS4 cells, Chinese hamster ovary cells (CHO), African green monkey kidney cells, human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NSO mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human B cells, human epithelial cells, human T cells, human dendritic cells, human macrophages, and human RAW cells. 264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

42. A cell culture comprising at least one recombinant cell and culture medium according to any one of claims 34-41.

43. A transgenic animal comprising: (a) a nucleic acid construct according to any one of claims 1-33; and / or (b) The recombinant cells according to any one of claims 34-41.

44. The transgenic animal according to claim 43, wherein the animal is a vertebrate or an invertebrate.

45. The transgenic animal according to claim 43, wherein the animal is an insect.

46. ​​The transgenic animal of claim 43, wherein the animal is a mammal.

47. The transgenic animal of claim 46, wherein the mammal is a non-human mammal.

48. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and: (a) a nucleic acid construct according to any one of claims 1-33; and / or (b) The recombinant cells according to any one of claims 34-41.

49. The pharmaceutical composition of claim 48, wherein the composition comprises a nucleic acid construct and a pharmaceutically acceptable excipient according to any one of claims 1-33.

50. The pharmaceutical composition according to any one of claims 48-49, wherein the composition is formulated together with a delivery medium into a delivery system, wherein the delivery system comprises liposomes, viral replicon particles (VRPs), lipid-based nanoparticles (LNPs), polymer nanoparticles, physiological buffers, microspheres, immunostimulatory complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof.

51. The pharmaceutical composition of claim 50, wherein the LNP delivery system comprises cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

52. The pharmaceutical composition of claim 51, wherein the lipids of the LNP delivery system are present in a mass ratio of lipid to RNA of about 100:1 to about 4:

1.

53. The pharmaceutical composition of claim 52, wherein the LNP delivery system comprises lipid-based nanoparticles with an average diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm or 25 nm.

54. The pharmaceutical composition of claim 52, wherein the LNP delivery system comprises lipid-based nanoparticles having an average diameter of about 1-1000 nm, about 1-1000 nm, about 1-500 nm, about 1-250 nm, about 25-200 nm, about 25-100 nm, about 35-75 nm, or about 25-60 nm.

55. The pharmaceutical composition according to any one of claims 48-54, wherein the composition is an immunogenic composition.

56. The pharmaceutical composition of claim 55, wherein the composition is formulated as a vaccine or adjuvant.

57. The pharmaceutical composition according to any one of claims 48-56, wherein the composition is formulated for use in one or more of the following routes of administration: intranasal administration, intrathecal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intra-articular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, and oral administration.

58. The pharmaceutical composition of claim 57, wherein the composition is formulated for intramuscular administration.

59. A method for inducing a pharmacodynamic effect in a subject with such need, the method comprising administering to the subject a composition comprising: a) The nucleic acid construct according to any one of claims 1-33; b) Recombinant cells according to any one of claims 34-41; and / or d) The pharmaceutical composition according to any one of claims 48-58.

60. The method of claim 59, wherein the pharmacodynamic effect includes one or more of the following: immunogenic effect, biomarker response, therapeutic effect, preventive effect, desired effect, undesired effect, adverse effect, and effect in a disease model.

61. The method of claim 60, wherein the pharmacodynamic effect includes prevention of viral infection and / or reduction of viral load in the subject.

62. The method of claim 60, wherein the pharmacodynamic effect includes prevention and / or reduction of the severity of the subject's disease.

63. The method according to any one of claims 60-62, wherein the pharmacodynamic effect includes inducing an immune response in the subject.

64. The method of claim 63, wherein the immune response comprises one or more of the following: an anti-EBV antibody response, a neutralizing antibody response, and an anti-EBV T cell response.

65. A method for preventing and / or treating a health condition in a subject with such need, the method comprising administering a composition to the subject preventively or therapeutically, the composition comprising: a) The nucleic acid construct according to any one of claims 1-33; b) Recombinant cells according to any one of claims 34-41; and / or c) The pharmaceutical composition according to any one of claims 48-58.

66. The method according to any one of claims 59-65, wherein the administered composition elicits an immune response in the subject.

67. The method according to any one of claims 59-66, wherein the administered composition results in an increase in the production of interferon by the subject.

68. The method according to any one of claims 59-67, wherein the subject has or is suspected of having EBV-related disease.

69. The method of claim 68, wherein the EBV-related disease is infectious mononucleosis (IM), an autoimmune disease, or cancer.

70. The method according to any one of claims 59-69, wherein the composition is administered to the subject alone as a single agent for prevention or treatment (single therapy) or as a first therapy in combination with at least one other therapy.

71. The method of claim 70, wherein the at least one additional therapy is selected from the group consisting of: chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery.

72. A kit for inducing pharmacodynamic effects, inducing immune responses, and / or for the prevention and / or treatment of health conditions, said kit comprising: a) The nucleic acid construct according to any one of claims 1-33; b) Recombinant cells according to any one of claims 34-41; and / or d) The pharmaceutical composition according to any one of claims 48-58, And a specification for performing the method according to any one of claims 59-71.

73. The kit according to claim 72, wherein the health condition is an EBV-related disease.

74. The kit according to claim 73, wherein the EBV-related disease is infectious mononucleosis (IM), an autoimmune disease, or cancer.

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