Epstein-barr virus vaccines

Abstract

To provide EBV ribonucleic acid vaccines as well as methods of using the vaccines and compositions comprising the vaccines.SOLUTION: Provided herein, in some embodiments, are Epstein-Barr virus (EBV) ribonucleic acid (RNA) (e.g., mRNA) vaccines (e.g., combination vaccines) that elicit potent neutralizing antibodies and robust T cell responses, inhibit the production of viral immunomodulatory factors, and / or prevent viral latency.SELECTED DRAWING: None

Description

Background Art

[0001] Epstein - Barr virus (EBV), also known as human herpesvirus 4, is one of the most common human viruses worldwide. Ninety - five percent of adults are infected with this virus. EBV most commonly spreads through body fluids (primarily saliva) and is a major cause of infectious mononucleosis (“mono”) and other diseases. Seventy - five percent of college students (18 - 22 years old) with a primary EBV infection will develop mono. Symptoms of EBV can include fatigue, fever, sore throat, swollen neck lymph nodes, spleen enlargement, liver enlargement, and rash. Many people are infected with EBV during childhood, but childhood symptoms are not distinguishable from other mild, short - term childhood illnesses. Typically, only teenagers and adults present the characteristic symptoms of EBV infection, and recovery is about 2 - 4 weeks, although some people may feel fatigued for weeks or months. Following EBV infection, the virus becomes latent and, in some cases, can be reactivated. People with a weakened immune system are more likely to develop symptoms when EBV is reactivated. Currently, there is no vaccine to prevent primary infection or disease.

Summary of the Invention

[0002] Epstein - Barr virus (EBV) ribonucleic acid (RNA) (e.g., mRNA) vaccines (e.g., combination vaccines) that elicit potent neutralizing antibodies and robust T - cell responses, inhibit the production of viral immunomodulatory factors, and / or prevent viral latency are provided in some embodiments herein. In some aspects, the EBV vaccine comprises RNA having an open reading frame (ORF) encoding an EBV antigen, and a therapeutically effective amount of intramuscular (IM) administration of the vaccine to a subject induces a neutralizing antibody titer and / or a T - cell immune response (e.g., an immune response of CD4+ T cells and / or CD8+ T cells) in the subject.

[0003] In some embodiments, the neutralizing antibody titer is at least 100 (e.g., at least 500 or at least 1000) NT after, for example, a single dose of the EBV RNA vaccine (e.g., a single dose of 10 μg to 200 μg). 50 In some embodiments, the neutralizing antibody titer is at least 100 (e.g., at least 500 or at least 1000) NT after a booster (second) dose of the EBV RNA vaccine. 50 It is.

[0004] In some embodiments, the neutralizing antibody titer is sufficient to reduce EBV infection of B cells by at least 50% (e.g., at least 60%, 70%, 80% or 90%) compared to the neutralizing antibody titer of unvaccinated control subjects or compared to the neutralizing antibody titer of subjects vaccinated with a live attenuated EBV vaccine, an inactivated EBV vaccine or a protein subunit EBV vaccine.

[0005] In some embodiments, the neutralizing antibody titer is induced in a subject after less than 3 (1 or 2) doses of the vaccine.

[0006] In some embodiments, the single dose of the EBV RNA vaccine is 10 μg to 100 μg.

[0007] In some embodiments, the neutralizing antibody titer and / or the T cell immune response are sufficient to reduce the rate of symptomatic infectious mononucleosis compared to the neutralizing antibody titer of unvaccinated control subjects.

[0008] In some embodiments, the neutralizing antibody titer and / or the T cell immune response are sufficient to reduce the rate of asymptomatic EBV infection compared to the neutralizing antibody titer of unvaccinated control subjects.

[0009] In some embodiments, the neutralizing antibody titer and / or the T cell immune response are sufficient to prevent EBV latency in a subject.

[0010] In some embodiments, the neutralizing antibody titer and / or the T cell immune response is sufficient to reduce chronic fatigue in a subject.

[0011] In some embodiments, the neutralizing antibody titer is sufficient to block the fusion of EBV with epithelial cells and / or B cells of a subject.

[0012] In some embodiments, the neutralizing antibody titer is induced within 20 days after a single 10 - 100 μg dose of the vaccine. In some embodiments, the neutralizing antibody titer is induced within 40 days after a second 10 - 100 μg dose of the vaccine.

[0013] In some embodiments, the ability of the vaccines of the disclosure to induce a neutralizing antibody response can be demonstrated by injecting the vaccine into an animal (e.g., a mouse or a non - human primate) and testing the ability of the serum from the animal to neutralize the ability of the virus to infect human B cells.

[0014] In some embodiments, the T cell immune response is CD4 + T cell immune response. In some embodiments, the T cell immune response is CD8 + T cell immune response. In some embodiments, the T cell immune response is CD4 + T cell immune response and CD8 + T cell immune response.

[0015] In some embodiments, after vaccination, EBV antigens are expressed on the surface of the subject's cells. In some embodiments, the ability of the vaccine to be expressed can be tested in a model system (e.g., a mouse or non - human primate model). In some embodiments, the ability of the vaccine to be expressed can be tested in vitro, for example, using human cells.

[0016] In some embodiments, a single 2 μg dose of the vaccine is about 100 NT 50Induce neutralizing antibody titers in mice. In some embodiments, a booster dose of 2 μg of the vaccine is about 1000 NT 50 Induce neutralizing antibody titers in mice.

[0017] In some embodiments, the EBV vaccine comprises one RNA having ORFs encoding two EBV antigens, or two RNAs each having an ORF encoding an EBV antigen.

[0018] In some embodiments, the vaccine comprises one RNA having ORFs encoding two (at least two) EBV antigens formulated in lipid nanoparticles. In some embodiments, the vaccine comprises two (at least two) RNAs each having an ORF encoding an EBV antigen, and the two RNAs are formulated in a single lipid nanoparticle. In some embodiments, the vaccine comprises two RNAs each having an ORF encoding an EBV antigen, and each RNA is formulated in a separate lipid nanoparticle.

[0019] In some embodiments, the EBV vaccine further comprises at least one (e.g., two, three, four, five or more) additional RNAs having ORFs encoding at least one (e.g., two, three, four, five or more) additional EBV antigens.

[0020] In some embodiments, the lipid nanoparticles comprise, in molar ratio, 20 - 60% ionizable cationic lipid, 5 - 25% non-cationic lipid, 25 - 55% sterol, and 0.5 - 15% PEG-modified lipid.

[0021] In some embodiments, the EBV antigen is selected from the group consisting of gp350, gH, gL, gB, gp42, LMP1, LMP2, EBNA1, and EBNA3.

[0022] In some embodiments, the EBV antigen is a gH-gL fusion, and gH is linked to gL via a linker (such as a GGGGS linker). In some embodiments, the GGGGS linker comprises three GGGGS motifs (SEQ ID NO: 224). In some embodiments, the GGGGS linker comprises four GGGGS motifs (SEQ ID NO: 225). In some embodiments, the EBV RNA comprises the nucleotide sequence of SEQ ID NO: 218. In some embodiments, the EBV RNA comprises the nucleotide sequence of SEQ ID NO: 221.

[0023] In some embodiments, EBV antigens include the EBV gp350 antigen, the EBV gH antigen, and the EBV gL antigen. In some embodiments, EBV antigens further include the EBV gp42 antigen and / or the gB antigen.

[0024] In some embodiments, the EBV gp350 antigen is a wild-type EBV gp350 antigen, a mutant EBV gp350 antigen, or a truncated EBV gp350 antigen.

[0025] In some embodiments, the EBV antigen is selected from the EBV antigens listed in the sequence listing.

[0026] In some embodiments, the EBV antigen(s) is fused to a scaffold moiety. In some embodiments, the scaffold moiety is selected from the group consisting of ferritin, encapsulin, lumazine synthase, hepatitis B surface antigen, and hepatitis B core antigen.

[0027] In some embodiments, the RNA includes messenger RNA (mRNA).

[0028] In some embodiments, the RNA further includes a 5’UTR. In some embodiments, the 5’UTR includes the sequence identified by SEQ ID NO: 1 or SEQ ID NO: 104.

[0029] In some embodiments, the RNA further comprises a 3'UTR. In some embodiments, the 3'UTR comprises the sequence identified by SEQ ID NO: 3 or SEQ ID NO: 106.

[0030] In some embodiments, the EBV antigen is fused to a signal peptide. In some embodiments, the signal peptide is a bovine prolactin signal peptide, optionally comprising SEQ ID NO: 115.

[0031] In some embodiments, the RNA is unmodified.

[0032] In some embodiments, the RNA comprises at least one modified nucleotide. In some embodiments, at least 80% (e.g., 90% or 100%) of the uracils in the ORF comprise a 1-methyl-pseudouridine modification.

[0033] Also provided in some aspects herein are methods that include administering to a subject a therapeutically effective amount of the disclosed EBV vaccine to induce in the subject a neutralizing antibody titer and / or a T cell immune response.

[0034] In some embodiments, the efficacy of the EBV vaccine is at least 80% (e.g., 85%, 90%, 95%, 98% or 100%) compared to unvaccinated control subjects.

[0035] In some embodiments, a detectable level of the EBV antigen is produced in the subject's serum 1 to 72 hours after administration of the vaccine.

[0036] In some embodiments, a neutralizing antibody titer of at least 100 (e.g., at least 500 or at least 1000) NU / ml is produced in the subject's serum 1 to 72 hours after administration of the vaccine.

[0037] In some embodiments, the therapeutically effective amount is a total dose of 20 μg to 200 μg (e.g., 50 μg to 100 μg).

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0039] Epstein-Barr virus (EBV) is a double-stranded DNA γ-herpesvirus that infects B cells and epithelial cells, causes infectious mononucleosis, and is associated with malignancies in both cell types in vivo (such as Burkitt lymphoma, Hodgkin lymphoma, and nasopharyngeal carcinoma). Approximately 95% of the population is infected with EBV by adulthood and carries EBV DNA throughout life. EBV is maintained in a latent state in infected B lymphocytes, with periodic reactivation of lytic replication occurring.

[0040] EBV (which spreads in all human populations) can be isolated in vitro via its ability to transform resting human B cells into permanent lymphoblastoid cell lines (LCLs) that express the EBV-encoded antigens EBNA1, 2, 3A, 3B, 3C, and LP, as well as latent membrane proteins (LMP) 1, 2A, and 2B. EBV isolates can be classified as type 1 or type 2 based on marked allelic polymorphisms within the EBNA2, 3A, 3B, and 3C genes, and into distinct strains based on more subtle sequence variations in the EBNA1, EBNA2, and LMP1 genes, as well as certain lytic cycle genes.

[0041] EBV has three glycoproteins (glycoprotein B (gB), gH, and gL) that form the core membrane fusion apparatus that mediates viral entry into cells. The gH protein and the gL protein associate to form a heterodimeric complex that is required for efficient membrane fusion and also participates in direct binding to epithelial cell receptors required for viral entry. EBV uses different pathways for the infection of epithelial cells and B lymphocytes. For both cell types, the minimal viral glycoprotein components that mediate membrane fusion have been identified. Like other herpesviruses, EBV uses the core viral entry glycoproteins (glycoprotein B (gB) and the gH / gL complex). For the infection of B lymphocytes, EBV requires an additional protein (gp42, which binds to host HLA class II molecules) that triggers the membrane fusion step. gp42 has multiple functional sites for interaction with another unknown binding ligand that can be engaged via gH / gL, HLA class II, and potentially a large hydrophobic pocket exposed on the surface. The gp42 protein binds to the gH / gL complex with nanomolar affinity via its N-terminal region, and this interaction can be recapitulated by a synthetic peptide of approximately 35 aa residues. EBV glycoprotein-mediated membrane fusion with epithelial cells does not require gp42 but only gB and gH / gL. Recent observations indicate that EBV gH / gL engages integrin αvβ6 and / or αvβ8 on epithelial cells to trigger membrane fusion and entry.

[0042] The EBV gp350 glycoprotein encoded by BLLF1 is important for efficient EBV infection of resting B cells. Gp350 is the most abundant viral protein in the viral envelope. This large protein is highly glycosylated and localizes to various intracellular compartments (cytoplasm, endoplasmic reticulum, Golgi, and plasma membrane) of replicating cells. EBV binds to primary B cells via interaction with CD21 (complement receptor 2 (CR2) via gp350). Multiple gp350 domains are thought to be involved in the formation of a stable complex with CD21, and one of them has been identified as the receptor-binding site (amino acids [aa] 142 - 161). This non-glycan domain is also recognized by the gp350-specific neutralizing antibody 72A.

[0043] The present disclosure is not limited to a particular strain of EBV. The strain of EBV used in the vaccine can be any strain of EBV.

[0044] The present disclosure provides an RNA (e.g., mRNA) vaccine against EBV infection (a vaccine that elicits a strong neutralizing antibody and a robust T cell response against EBV antigens, inhibits the production of viral immunomodulatory factors, and / or prevents viral latency).

[0045] In some embodiments, the vaccines disclosed herein are used therapeutically (i.e., after infection by EBV) (to treat the infection). In some embodiments, the vaccines of the present disclosure can be used to prevent or reduce the frequency of Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, nasopharyngeal cancer, post-transplant lymphoproliferative disorder, diffuse large B cell lymphoma, and / or NK / T cell lymphoma.

[0046] The EBV RNA vaccines described herein are superior to current vaccines in several respects. For example, the lipid nanoparticle (LNP) delivery system used herein increases the efficacy of RNA vaccines compared to other formulations (including the protamine-based approaches described in the literature). The use of this LNP delivery system enables the effective delivery of chemically modified or unmodified RNA vaccines without the requirement for additional adjuvants to produce therapeutic outcomes (e.g., the production of neutralizing antibody titers and / or T cell responses). In some embodiments, the EBV RNA vaccines disclosed herein are at least 10-fold, 20-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1,000-fold superior to conventional vaccines when administered intramuscularly (IM) or intradermally (ID). These results can be achieved even when significantly lower doses of RNA (e.g., mRNA) are administered compared to the RNA doses used in other classes of lipid-based formulations.

[0047] The LNPs used in the studies described herein have been previously used to deliver siRNA in various animal models in addition to humans. Considering the observations made in connection with siRNA delivery by LNP formulations, the fact that LNPs are useful for vaccines, particularly when the generation of immunity to an antigen is difficult as in the case of EBV, is quite unexpected. It has been observed that the therapeutic delivery of siRNA formulated in LNPs causes an undesired inflammatory response associated with a transient IgM response, typically leading to a reduction in antigen production and an impairment of the immune response. In contrast to the findings observed with siRNA, the LNP-mRNA formulations of the present disclosure are demonstrated herein to produce enhanced IgG levels (sufficient for prophylactic and therapeutic methods, rather than a transient IgM response).

[0048] Exemplary Epstein-Barr virus (EBV) antigens An antigen is a protein capable of inducing an immune response (e.g., causing the immune system to produce antibodies against the antigen). As used herein, the term "antigen," unless otherwise specified, encompasses immunogenic proteins and immunogenic fragments (immunogenic fragments that induce (or are capable of inducing) an immune response against EBV). It should be understood that the term "protein" encompasses peptides and the term "antigen" encompasses antigenic fragments.

[0049] Many different antigens are associated with EBV. An EBV vaccine as provided herein comprises at least one (one or more) ribonucleic acid (RNA, e.g., mRNA) having an open reading frame encoding at least one EBV antigen. Non-limiting examples of EBV antigens are provided below.

[0050] Exemplary EBV antigens are provided in the sequence listings elsewhere in this specification. For example, the antigen can be encoded by any one of the sequences described in SEQ ID NOs: 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, and / or 210 (thus the RNA can contain or consist of it). In some embodiments, the sequence further comprises a 5' cap (e.g., 7mG(5')ppp(5')NlmpNp), a polyA tail, or both a 5' cap and a polyA tail.

[0051] It should be understood that the EBV vaccines of the present disclosure may include any of the RNA open reading frames (ORFs), or may encode any of the protein ORFs described herein with or without a signal sequence. It should also be understood that the EBV vaccines of the present disclosure may include any 5' untranslated region (UTR) and / or any 3' UTR. Exemplary UTR sequences are provided in the sequence listing (e.g., SEQ ID NOs: 1, 3, 104, and 106), but other UTR sequences (e.g., of the prior art) may be used or they may replace any of the UTR sequences described herein. The UTR may also be omitted from the vaccine constructs provided herein.

[0052] EBV entry into B cells is initiated by the attachment of the glycoprotein gp350 to the complement receptor 2 (CR2). A complex of three glycoproteins (gH, gL, and gp42) is subsequently required for penetration. gp42 binds to HLA class II, which functions as an entry mediator or coreceptor, and by analogy with other herpesviruses, gH is then thought to be involved in virus-cell fusion. Virus entry into epithelial cells is different. It can be initiated by attachment by an unknown glycoprotein in the absence of CR2. There is no interaction between gp42 and HLA class II; instead, a distinct complex of just the two glycoproteins gH and gL interacts with a novel entry mediator.

[0053] The EBV gH-gL complex includes three glycoproteins: gp85 (a gH homolog, which is the product of the BXLF2 open reading frame (ORF)); gp25 (a gL homolog, which is the product of the BKRF2 ORF); and gp42 (which is the product of the BZLF2 ORF). The complex acts in many respects similarly to its counterpart in other herpesviruses. The glycoprotein gH is dependent on gL for proper processing and transport, and the complex as a whole was implicated as being important for the ability of the virus to fuse with the cell membrane and penetrate into the cytoplasm.

[0054] The gp350 glycoprotein encoded by BLLF1 is important for efficient Epstein - Barr virus (EBV) infection of resting B cells.

[0055] The major EBV glycoprotein gp350 mediates EBV docking on B cells by binding to the type 2 receptor (CR2) (Nemerow et al., J of Virol. (61):1416 - 1420(1987); Szakonyi et al., Nat Struct Mol Biol. (13):996 - 1001(2006)). Due to alternative splicing, BLLF1 encodes gp350 and gp220, which are glycosylated and are approximately 350 kilodaltons and 220 kilodaltons in molecular weight respectively (Beisel et al., J Virol. (54):665 - 674(1985); Hummel et al., J Virol. (49):413 - 417(1984)). In some embodiments, the EBV gp350 antigen comprises a sequence identified by SEQ ID NO: 81, 204, 185, 182, 207 or 208.

[0056] After EBV docking, EBV fuses with the host cell plasma membrane using a glycoprotein complex. The core EBV membrane fusion apparatus for entry into B cells and epithelial cells comprises glycoprotein B (gB), glycoprotein H (gH) and glycoprotein L (gL) (Hutt - Fletcher et al., J Virol. (81):7825 - 7832(2007)).

[0057] gB is a type I single-pass transmembrane protein also known as gp110 and is encoded by the BALF4 open reading frame (ORF) (Herrold et al., J of Virol. (70):2049-2054 (1996); Haan et al., Virology (290):106-114 (2001)); McShane et al., Proc Natl Acad Sci USA. (101):17474-17479 (2004)). In some embodiments, the EBV gB antigen comprises the sequence identified by SEQ ID NO: 209.

[0058] gH (also known as gp85) is a type I transmembrane protein encoded by the open reading frame (ORF) of the BXLF2 gene (Heineman et al., J Virol. (62):1101-1107 (1988)); Oba et al., J Virol. (62):1108-1114 (1988)). In some embodiments, the EBV gH antigen comprises the sequence identified by SEQ ID NO: 187.

[0059] gL (also known as gp25) encoded by the BKRF2 ORF is required for proper folding and localization of gH (Li et al., J Virol. (69):3987-3994 (1995); Yaswen et al., Virology. (195):387-396 (1993)). Thus, gH and gL often function as a complex to mediate virus fusion, and this complex has been crystallized (Matsurra et al., Proc Natl Acad Sci USA. (107):22641-2264 (2010)). In some embodiments, the EBV gL antigen comprises the sequence identified by SEQ ID NO: 188.

[0060] In addition to the core membrane fusion apparatus, EBV entry into B cells requires gp42, which is encoded by the BZLF2 ORF (Kirschner et al., J. Virol. (80):9444 - 54(2006); Wang et al., J. Virol., (72):5552 - 5558(1998); Silva et al., J. Virol. (78):5946 - 5956(2004); Li et al. J. Virol., (69):3987 - 3994(1995)). EBV gp42 mediates the fusion of B cells and the virus by binding to MHC class II molecules (Mullen et al., Molecular Cell. (9):375 - 385(2002); Haan et al. J Virol. (74):2451 - 4(2000)). In some embodiments, the EBV gp42 antigen comprises a sequence identified by SEQ ID NO:189.

[0061] Latent membrane protein 1 (LMP1) is a six - transmembrane domain protein that enhances the immortalization of resting B cells and helps protect EBV - infected B cells from apoptosis (Hennessy et al., Proc Natl. Acad. Sci USA. (81):7207 - 11(1984); Kaye et al., Proc Natl Acad Sci USA. (90):9150 - 9154(1993); Henderson et al., Cell(65):1107 - 1115(1991)). A number of signaling pathways are activated by LMP1, including signaling of the tumor necrosis factor receptor family and DNA synthesis (Peng et al. Oncogene. (7):1775 - 1782; Masialos et al., Cell. (80):389 - 399(1995); Li et al., J Biomed Sci. (10):490 - 504(2003)). Furthermore, LMP1 signaling can up - regulate the expression of the anti - apoptotic Bcl - 2 cancer gene in B cells (Rowe et al., J Virol. (68):5602 - 12(1994)). In some embodiments, the EBV LMP1 antigen comprises a sequence identified by SEQ ID NO:179.

[0062] Similar to LMP1, latent membrane protein 2 (LMP2) is a transmembrane protein encoded by EBV that is often expressed in latently infected cells. There are two isoforms of LMP2 (LMP2A and LMP2B) (Laux et al., EMBO J. (7):769-74 (1988); Longnecker et al., J Virol. (64):2319-26 (1990)). LMP2A is involved in the maintenance of EBV latency. For example, LMP2A can exclude the B cell receptor (BCR) from lipid rafts and prevent lytic induction (Dykstra et al., Immunity. (14):57-67 (2001)). LMP2A can also activate the phosphatidylinositol 3-kinase (PI3-K) / Akt pathway to enhance cell survival (Scholle et al., J Virol. (74):10681-10689 (2000); Swart et al. J Virol. (74):10838-10845 (2000); Fukuda et al., J.Virol. (78):1697-16705 (2004)). The LMP2B protein generally lacks 119 amino-terminal amino acids compared to LMP2A and is involved in epithelial cell spreading and motility (Allen et al., J Virol. (79):1789-1802 (2005)). In some embodiments, the EBV LMP2 antigen comprises the sequence identified by SEQ ID NO: 181.

[0063] Epstein-Barr nuclear antigens (ENBAs) that assist in establishing latent infection include EBNA1, EBNA2, EBNA3A, and EBNA3C. EBNA1, encoded by BKRF1, enhances viral DNA replication, episomal maintenance, and episomal distribution (Rawlins et al., Cell (42):859-68 (1985); Hung et al., Proc Natl Acad Sciences USA (98):1865-1870 (2001)). In particular, EBNA1 can bind to the repeat family and dyad symmetry elements of the latent origin of replication oriP. In some embodiments, the EBV EBNA1 antigen comprises the sequence identified by SEQ ID NO: 178.

[0064] The EBNA3 family has three members (EBNA3A, EBNA3B, and EBNA3C). EBNA3 regulates transcription by binding to RBPJ, which is a transcriptional regulator in the Notch signaling pathway (Zhao et al., J Virol. (70):4228-4236 (1996); Robertson et al., J Virol. (69):3108-3116 (1995); Robertson et al. J Virol. (70):3068-3074 (1996)). In particular, EBNA3A and EBNA3C have been shown to be required for EBV-mediated transformation of B cells (Tomkinson et al. J Virol. (67):2014-25 (1993)). In some embodiments, the EBV EBNA3A antigen comprises the sequence identified by SEQ ID NO: 177.

[0065] Nucleic acid The EBV vaccine of the present disclosure comprises at least one (one or more) ribonucleic acid (RNA) having an open reading frame encoding at least one EBV antigen. In some embodiments, the RNA is a messenger RNA (mRNA) having an open reading frame encoding at least one EBV antigen. In some embodiments, the RNA (e.g., mRNA) further comprises a (at least one) 5’UTR, 3’UTR, polyA tail, and / or 5’ cap.

[0066] Nucleic acids include polymers of nucleotides (nucleotide monomers), also referred to as polynucleotides. Nucleic acids can be, or can include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid, (LNA, including LNA having a β-D-ribo configuration, α-LNA (a diastereoisomer of LNA) having an α-L-ribo configuration, 2’-amino-LNA having a 2’-amino functionalization, and 2’-amino-α-LNA having a 2’-amino functional group), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), and / or chimeras and / or combinations thereof.

[0067] Messenger RNA (mRNA) is any ribonucleic acid that encodes a (at least one) protein (a polymer of naturally occurring, non-naturally occurring, or modified amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. Those skilled in the art will recognize that the nucleic acid sequences described in this application will list “T” in a representative DNA sequence, except where specifically indicated, but “T” will be replaced by “U” when the sequence represents RNA (e.g., mRNA). Thus, any of the DNAs disclosed and identified herein by a particular sequence identifier also discloses the corresponding RNA (e.g., mRNA) sequence complementary to the DNA, where “T” in the DNA sequence is replaced by “U” in each case.

[0068] An open reading frame (ORF) is a continuous stretch of DNA or RNA that starts with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). An ORF typically encodes a protein. The sequences disclosed herein may further include additional elements (e.g., 5’UTR and 3’UTR), but it will be understood that these elements, unlike the ORF, do not necessarily have to be present in the vaccines of the present disclosure.

[0069] Variant In some embodiments, the RNA of the present disclosure encodes an EBV antigen variant. An antigen or other polypeptide variant refers to a molecule that is different in amino acid sequence from a wild-type sequence, a native sequence or a reference sequence. An antigen / polypeptide variant may retain substitutions, deletions and / or insertions at certain positions within the amino acid sequence as compared to the native or reference sequence. Typically, a variant retains at least 50% identity to the wild-type sequence, native sequence or reference sequence. In some embodiments, the variant shares at least 80% or at least 90% identity with the wild-type sequence, native sequence or reference sequence.

[0070] The variant antigens / polypeptides encoded by the nucleic acids of the present disclosure can contain amino acid changes that confer any of a number of desired properties, such as those that enhance their immunogenicity, those that enhance their expression, and / or those that improve their stability or PK / PD properties in a subject. The variant antigens / polypeptides are made using routine mutagenesis techniques and can be assayed as needed to determine whether they retain the desired properties. Assays for determining expression levels and immunogenicity are well known in the art, and exemplary such assays are described in the Examples section. Similarly, the PK / PD properties of protein variants can be measured using techniques recognized in the art, such as by determining the expression of the antigen over time in a vaccinated subject and / or by looking at the durability of the induced immune response. The stability of the protein(s) encoded by the variant nucleic acids can be measured by assays of thermal stability or stability upon urea denaturation, or can be measured using in silico predictions. Methods for such experiments and in silico determinations are known in the art.

[0071] In some embodiments, the EBV vaccine comprises an mRNA ORF having a nucleotide sequence identified by any one of the sequences provided herein (see, e.g., the Sequence Listing), or having a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a nucleotide sequence identified by any one of the sequences provided herein.

[0072] The term "identity" refers to the relationship between the sequences of two or more polypeptides (e.g., antigens) or polynucleotides (nucleic acids) as determined by comparison of the sequences. Identity also refers to the degree of sequence correlation between or within sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity is measured as the percentage of identical matches between the smaller of two or more sequences, treated using gap alignment (if any), by a particular mathematical model or computer program (e.g., an "algorithm"). The identity of related antigens or nucleic acids can be readily calculated by known methods. "Percent (%) identity", when applied to the sequence of a polypeptide or polynucleotide, is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical to residues in the amino acid sequence or nucleic acid sequence of a second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent identity. Methods and computer programs for alignment are well known in the art. It is understood that identity depends on the calculation of percent identity, but the values can differ due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide (e.g., antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity to that particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters known to those of skill in the art described herein. Such tools for alignment include those of the BLAST package of programs (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402).Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197). A common global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453). More recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which produces global alignments of nucleotide and protein sequences faster than perhaps any other optimal global alignment method (including the Needleman-Wunsch algorithm).

[0073] Accordingly, with respect to a reference sequence (especially a polypeptide (e.g., antigen) sequence disclosed herein), polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions as well as covalent modifications are included within the scope of the present disclosure. For example, a sequence tag or an amino acid (such as one or more lysines) can be added to a peptide sequence (e.g., at the N-terminus or C-terminus). The sequence tag can be used for detection, purification or localization of the peptide. Lysine can be used to increase peptide solubility or enable biotinylation. Alternatively, amino acid residues located in the carboxy-terminal region and amino-terminal region of the amino acid sequence of a peptide or protein can optionally be deleted to provide a truncated sequence. Alternatively, certain amino acids (e.g., C-terminal or N-terminal residues) can be deleted depending on the use of the sequence (e.g., whether it is soluble or linked to a solid support, expression of the sequence as part of a larger sequence). In some embodiments, sequences for signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (e.g., foldon regions, etc.) and the like (or those encoding them) can be replaced by alternative sequences that achieve the same or similar functions. In some embodiments, cavities in the core of a protein can be filled, for example, by introduction of larger amino acids to improve stability. In other embodiments, buried hydrogen bond networks can be replaced by hydrophobic residues to improve stability. In yet other embodiments, glycosylation sites can be removed and replaced by appropriate residues. Such sequences can be readily identified by those skilled in the art. It is also to be understood that some of the sequences provided herein can contain sequence tags or terminal peptide sequences (e.g., at the N-terminus or C-terminus) that can be deleted, for example, prior to use in the preparation of an RNA (e.g., mRNA) vaccine.

[0074] As will be recognized by those skilled in the art, protein fragments, functional protein domains and homologous proteins are also determined to be within the scope of the EBV antigens of interest. For example, any protein fragment of a reference protein (meaning a polypeptide sequence that is at least 1 amino acid residue shorter than the reference antigen sequence but otherwise identical) is provided herein, provided that the fragment is immunogenic and confers a protective immune response against the EBV pathogen. In addition to variants that are identical to but shorter than the reference protein, in some embodiments, the antigen comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations as shown in any of the sequences provided or referenced herein. The antigen / antigenic polypeptide can range from about 4, 6 or 8 amino acids to the length of the full-length protein.

[0075] Stabilizing element Naturally occurring eukaryotic mRNA molecules can contain stabilizing elements, including, but not limited to, untranslated regions (UTRs) at the 5' end (5'UTR) and / or UTRs at the 3' end (3'UTR), as well as other structural features (such as the 5' cap structure or the 3' poly(A) tail). Both the 5'UTR and the 3'UTR are typically transcribed from genomic DNA and are elements of the immature mRNA. Characteristic structural features of mature mRNA (such as the 5' cap and the 3' poly(A) tail) are usually added to the transcribed (immature) mRNA during mRNA processing.

[0076] In some embodiments, the vaccine comprises at least one RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide having at least one modification, includes at least one 5′ end cap, and is formulated within lipid nanoparticles. 5′ capping of the polynucleotide can be accomplished simultaneously during an in vitro transcription reaction by generating a 5′ guanosine cap structure using the following chemical RNA cap analogs according to the manufacturer's protocol: 3′-O-Me-m7G(5′)ppp(5′)G [ARCA cap]; G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (New England BioLabs, Ipswich, MA). 5′ capping of the modified RNA can be accomplished by using a vaccinia virus capping enzyme post-transcription to generate a “cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs, Ipswich, MA). A cap 1 structure is generated using both a vaccinia virus capping enzyme and a 2′-O methyltransferase, and m7G(5′)ppp(5′)G-2′-O-methyl can be generated. A cap 2 structure can be generated from the cap 1 structure, followed by 2′-O methylation of the third nucleotide from the 5′ end using a 2′-O methyltransferase. A cap 3 structure can be generated from the cap 2 structure, followed by 2′-O methylation of the fourth nucleotide from the 5′ end using a 2′-O methyltransferase. The enzymes can be of recombinant origin.

[0077] The 3′ poly(A) tail is typically a stretch of adenine nucleotides added to the 3′ end of the transcribed mRNA. In some cases, it can contain up to about 400 adenine nucleotides. In some embodiments, the length of the 3′ poly(A) tail can be an essential element with respect to the stability of an individual mRNA.

[0078] In some embodiments, the EBV RNA vaccine may contain one or more stabilizing elements. Examples of stabilizing elements may include histone stem-loops. A stem-loop binding protein (SLBP) of the 32 kDa protein has been identified. SLBP associates with the histone stem-loop at the 3’ end of the histone message in both the nucleus and the cytoplasm. The expression level of SLBP is regulated by the cell cycle and reaches a peak during the S phase when the level of histone mRNA also increases. This protein has been shown to be essential for the efficient 3’ end processing of histone pre-mRNA by U7 snRNP. SLBP continues to associate with the stem-loop after processing and then stimulates the translation of mature histone mRNA into histone protein in the cytoplasm. The RNA binding domain of SLBP is conserved throughout metazoans and protozoans, and its binding to the histone stem-loop depends on the structure of the loop. The minimal binding site contains at least 3 nucleotides 5’ to the stem-loop and 2 nucleotides 3’ to it.

[0079] In some embodiments, the EBV RNA vaccine contains a coding region, at least one histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally promotes the expression level of the encoded protein. The encoded protein is, in some embodiments, not a histone protein, a reporter protein (e.g., luciferase, GFP, EGFP, β-galactosidase, EGFP), or a marker or selectable protein (e.g., α-globin, galactokinase, and xanthine:guanine phosphoribosyltransferase (GPT)).

[0080] In some embodiments, the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem loop acts synergistically to increase protein expression beyond the levels observed by either of the individual elements, even if both represent native and alternative mechanisms. The synergistic effect of the combination of poly(A) and at least one histone stem loop is independent of the order of the elements or the length of the poly(A) sequence.

[0081] In some embodiments, the EBV RNA vaccine does not contain a histone downstream element (HDE). The “histone downstream element” (HDE) refers to an approximately 15-20 nucleotide purine-rich polynucleotide stretch on the 3′ side of a naturally occurring stem loop, which represents a binding site for U7 snRNA (involved in the processing of histone pre-mRNA to mature histone mRNA). In some embodiments, the nucleic acid does not contain an intron.

[0082] In some embodiments, the EBV RNA vaccine contains or does not contain an enhancer sequence and / or a promoter sequence, which may be modified or unmodified, or activated or inactivated. In some embodiments, a histone stem loop generally comprises intramolecular base pairing of two adjacent partial or fully reverse complementary sequences separated by a spacer (consisting of short sequences) that forms a loop in the structure. The unpaired loop region typically cannot base pair with either of the stem loop elements. It is an important component of many RNA secondary structures and thus occurs more frequently in RNA, but can also be present in single-stranded DNA. The stability of the stem loop structure generally depends on the length, the number of mismatches or bulges, and the base composition of the paired region. In some embodiments, wobble base pairs (non-Watson-Crick base pairs) can occur. In some embodiments, at least one histone stem loop sequence comprises 15-45 nucleotides in length.

[0083] In some embodiments, the EBV RNA vaccine can remove one or more AU-rich sequences. These sequences (sometimes referred to as AURES) are destabilizing sequences found in the 3’UTR. AURES can be removed from the RNA vaccine. Alternatively, AURES may remain in the RNA vaccine.

[0084] Signal peptide In some embodiments, the EBV vaccine comprises an RNA having an ORF encoding a signal peptide fused to an EBV antigen. The signal peptide (comprising 15-60 amino acids at the N-terminus of the protein) is typically required for translocation across membranes in the secretory pathway and thus generally controls the entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) directs ribosomes to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across the ER membrane for processing. ER processing produces a mature protein, where the signal peptide is typically cleaved from the precursor protein by the host cell's ER-resident signal peptidase, or they remain uncleaved and function as a membrane anchor. The signal peptide can also facilitate the targeting of the protein to the cell membrane.

[0085] The signal peptide can have a length of 15 to 60 amino acids. For example, the signal peptide can have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acids. In some embodiments, the signal peptide has a length of 20 to 60, 25 to 60, 30 to 60, 35 to 60, 40 to 60, 45 to 60, 50 to 60, 55 to 60, 15 to 55, 20 to 55, 25 to 55, 30 to 55, 35 to 55, 40 to 55, 45 to 55, 50 to 55, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 15 to 45, 20 to 45, 25 to 45, 30 to 45, 35 to 45, 40 to 45, 15 to 40, 20 to 40, 25 to 40, 30 to 40, 35 to 40, 15 to 35, 20 to 35, 25 to 35, 30 to 35, 15 to 30, 20 to 30, 25 to 30, 15 to 25, 20 to 25 or 15 to 20 amino acids.

[0086] Signal peptides from heterologous genes (which naturally regulate the expression of genes other than EBV antigens) are known in the art, can be tested for desired properties, and then incorporated into the nucleic acids of the present disclosure. In some embodiments, the signal peptide is the bovine prolactin signal peptide. For example, the bovine prolactin signal peptide can include the sequence MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG (SEQ ID NO: 115). Other signal peptide sequences can also be used. For example, the signal peptide can include one of the following sequences. MDWTWILFLVAAATRVHS (SEQ ID NO: 116); METPAQLLFLLLLWLPDTTG (SEQ ID NO: 117); MLGSNSGQRVVFTILLLLVAPAYS (SEQ ID NO: 118); MKCLLYLAFLFIGVNCA (SEQ ID NO: 119); MWLVSLAIVTACAGA (SEQ ID NO: 120).

[0087] Fusion protein In some embodiments, the EBV RNA vaccine of the present disclosure comprises RNA encoding an antigenic fusion protein. Thus, the encoded antigen(s) can include two or more proteins (e.g., proteins and / or protein fragments) linked together. Alternatively, the protein to which the protein antigen is fused does not enhance a strong immune response to itself, but rather enhances a strong immune response to the EBV antigen. The antigenic fusion protein retains, in some embodiments, the functional properties from each of the original proteins.

[0088] Scaffold portion RNA (e.g., mRNA) vaccines as provided herein encode, in some embodiments, a fusion protein comprising an EBV antigen linked to a scaffold portion. In some embodiments, such a scaffold portion imparts desired properties to the antigen encoded by the nucleic acids of the present disclosure. For example, the scaffold protein can improve the immunogenicity of the antigen by causing, for example, altering the structure of the antigen, altering the uptake and processing of the antigen, and / or binding the antigen to a binding partner.

[0089] In some embodiments, the scaffold portion is a protein that can self-assemble into protein nanoparticles with a diameter of 10 to 150 nm (a size range highly suitable for optimal interaction with various cells of the immune system), which are highly symmetric, stable, and structurally organized. In some embodiments, viral proteins or virus-like particles can be used to form stable nanoparticle structures. Examples of such viral proteins are known in the art. For example, in some embodiments, the scaffold portion is hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles with an average diameter of about 22 nm, which lack nucleic acid and are thus non-infectious (Lopez-Sagaseta, J. et al. Computational and Structural Biotechnology Journal 14 (2016) 58 - 68). In some embodiments, the scaffold portion is hepatitis B core antigen (HBcAg), which self-assembles into particles with a diameter of 24 - 31 nm and resembles the viral core obtained from HBV-infected human liver. The produced HBcAg self-assembles into two different classes of nanoparticles with diameters of 300 Å and 360 Å (corresponding to 180 or 240 protomers). In some embodiments, the EBV antigen is fused to HBsAG or HBcAG to promote the self-assembly of nanoparticles presenting the EBV antigen.

[0090] In another embodiment, a platform of bacterial proteins can be used. Non-limiting examples of these self-assembling proteins include ferritin, lumazine, and encapsulin.

[0091] Ferritin is a protein whose main function is intracellular iron storage. Ferritin consists of 24 subunits, each composed of four α-helix bundles, which self-assemble into a quaternary structure with octahedral symmetry (Cho K.J. et al. J Mol Biol. 2009;390:83-98). Multiple high-resolution structures of ferritin have been determined, and it has been confirmed that the ferritin of Helicobacter pylori is composed of 24 identical protomers. However, in animals, there are light and heavy chains of ferritin that can assemble alone or combine in different ratios into 24-subunit particles (Granier T. et al. J Biol Inorg Chem. 2003;8:105-111; Lawson D.M. et al. Nature. 1991;349:541-544). Ferritin self-assembles into nanoparticles with strong thermal and chemical stability. Therefore, ferritin nanoparticles are well-suited for carrying and exposing antigens.

[0092] Luciferase synthase (LS) is also well-suited as a nanoparticle platform for antigen presentation. LS (which is involved in the second-to-last catalytic step in the biosynthesis of riboflavin) is an enzyme present in a wide variety of organisms, including archaea, bacteria, fungi, plants, and eubacteria (Weber S.E. Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014). The LS monomer is 150 amino acids long and consists of tandem α-helices flanking a β-sheet on both sides. A number of different quaternary structures have been reported for LS, exemplifying its morphological versatility, from homopentamers to symmetric assemblies of 12 pentamers forming a 150 Å diameter capsid. LS cages with over 100 subunits have even been described (Zhang X. et al. J Mol Biol. 2006;362:753-770).

[0093] Encapsulin (a novel protein cage nanoparticle isolated from the hyperthermophilic Thermotoga maritima) has also been used as a platform to display antigens on the surface of self-assembling nanoparticles. Encapsulin assembles 60 copies of the same 31 kDa monomer to form a thin icosahedral (T = 1) symmetric cage structure with internal and external diameters of 20 nm and 24 nm, respectively (Sutter M. et al. Nat Struct Mol Biol. 2008, 15:939-947). The exact function of encapsulin in T. maritima is not yet clearly understood, but its crystal structure has recently been resolved and its function has been proposed as an intracellular compartment that encapsulates proteins (such as DyP (dye-decolorizing peroxidase) and Flp (ferritin-like protein), which are involved in the oxidative stress response) (Rahmanpour R. et al. FEBS J. 2013, 280:2097-2104).

[0094] Linker and cleavable peptide In some embodiments, the mRNA of the present disclosure encodes two or more polypeptides, which are referred to herein as fusion proteins. In some embodiments, the mRNA further encodes a linker located between at least one or each domain of the fusion protein. The linker can be, for example, a cleavable linker or a protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of an F2A linker, a P2A linker, a T2A linker, an E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, called 2A peptides, has been described in the art (see, for example, Kim, J.H. et al. (2011) PLoS ONE 6: e18556). In some embodiments, the linker is an F2A linker. In some embodiments, the linker is a GGGS linker or a GGGGS linker, and examples include one or more (e.g., 1, 2, 3, 4, or more) repeating sequences of GGGS (SEQ ID NO: 226) or GGGGS (SEQ ID NO: 227) (e.g., GGGGS GGGGS GGGGS (SEQ ID NO: 224) and / or GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 225)). In some embodiments, the fusion protein contains three domains together with intervening linkers and has the structure: domain-linker-domain-linker-domain.

[0095] Cleavable linkers known in the art can be used in connection with the present disclosure. Exemplary such linkers include an F2A linker, a T2A linker, a P2A linker, an E2A linker (see, for example, WO2017 / 127750). Those skilled in the art will recognize that other linkers recognized in the art may be suitable for use in the constructs of the present disclosure (e.g., encoded by the nucleic acids of the present disclosure). Those skilled in the art will similarly recognize that other polycistronic constructs (mRNAs encoding two or more antigens / polypeptides separately within the same molecule) may be suitable for use as provided herein.

[0096] Array Optimization In some embodiments, the ORFs encoding the antigens of the present disclosure are codon-optimized. Codon optimization methods are known in the art. For example, any one or more ORFs of the sequences provided herein can be codon-optimized. Codon optimization can be used in some embodiments to match codon frequencies in the target and host organisms to ensure proper folding; bias the GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeat codons or base runs that can worsen gene construction or expression; customize transcriptional and translational regulatory regions; insert or remove protein trafficking sequences; remove / add post-translational modification sites in the encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust the translation rate to allow proper folding of various domains of the protein; or reduce or eliminate problematic secondary structures within the polynucleotide. Tools, algorithms and services for codon optimization are known in the art and include, by way of non-limiting example, GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or services from in-house methods. In some embodiments, the open reading frame (ORF) sequences are optimized using an optimization algorithm.

[0097] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity to the open reading frame (ORF) of a naturally occurring or wild-type sequence (e.g., the sequence of a naturally occurring or wild-type mRNA encoding an EBV antigen). In some embodiments, the codon-optimized sequence shares less than 90% sequence identity to a naturally occurring or wild-type sequence (e.g., the sequence of a naturally occurring or wild-type mRNA encoding an EBV antigen). In some embodiments, the codon-optimized sequence shares less than 85% sequence identity to a naturally occurring or wild-type sequence (e.g., the sequence of a naturally occurring or wild-type mRNA encoding an EBV antigen). In some embodiments, the codon-optimized sequence shares less than 80% sequence identity to a naturally occurring or wild-type sequence (e.g., the sequence of a naturally occurring or wild-type mRNA encoding an EBV antigen). In some embodiments, the codon-optimized sequence shares less than 75% sequence identity to a naturally occurring or wild-type sequence (e.g., the sequence of a naturally occurring or wild-type mRNA encoding an EBV antigen).

[0098] In some embodiments, the codon-optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally occurring or wild-type sequence (e.g., the sequence of a naturally occurring or wild-type mRNA encoding an EBV antigen). In some embodiments, the codon-optimized sequence shares between 65% and 75% or about 80% sequence identity to a naturally occurring or wild-type sequence (e.g., the sequence of a naturally occurring or wild-type mRNA encoding an EBV antigen).

[0099] In some embodiments, the codon-optimized sequence encodes an antigen that is as immunogenic as or more immunogenic (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100% or at least 200% more) than the EBV antigen encoded by the non-codon-optimized sequence.

[0100] When transfected into mammalian host cells, the modified mRNA has stability between 12 and 18 hours or for 18 hours or more (e.g., 24, 36, 48, 60, 72 or more than 72 hours) and can be expressed by mammalian host cells.

[0101] In some embodiments, the codon-optimized RNA can have an enhanced G / C level. The G / C content of a nucleic acid molecule (e.g., mRNA) can affect the stability of the RNA. RNA having an increased amount of guanine (G) and / or cytosine (C) residues can be functionally more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO02 / 098443 discloses a pharmaceutical composition containing mRNA stabilized by sequence modification in the translated region. Due to the degeneracy of the genetic code, this modification works by substituting existing codons with ones that further enhance RNA stability without changing the resulting amino acids. This approach is not limited to the coding region of the RNA.

[0102] Unmodified nucleotides chemically In some embodiments, at least one RNA (e.g., mRNA) of the EBV vaccines of the present disclosure is not chemically modified and contains standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides of the present disclosure contain standard nucleoside residues (such as those present in the transcribed RNA (e.g., A, G, C, or U, etc.)). In some embodiments, the nucleotides and nucleosides of the present disclosure contain standard deoxyribonucleosides (such as those present in DNA (e.g., dA, dG, dC, or dT, etc.)).

[0103] Chemical modification In some embodiments, the EBV RNA vaccines of the present disclosure comprise at least one nucleic acid (e.g., RNA) having an open reading frame encoding at least one EBV antigen, wherein the nucleic acid contains standard (unmodified) or modified nucleotides and / or nucleosides, as is known in the art. In some embodiments, the nucleotides and nucleosides of the present disclosure contain modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. As recognized in the art, such modifications can include those on the sugar, backbone, or nucleobase moiety of the nucleotide and / or nucleoside.

[0104] In some embodiments, the naturally occurring modified nucleotides or nucleotides of the present disclosure are those generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

[0105] In some embodiments, the non-naturally occurring modified nucleotides or nucleosides of the present disclosure are those generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found inter alia in published U.S. applications PCT / US2012 / 058519; PCT / US2013 / 075177; PCT / US2014 / 058897; PCT / US2014 / 058891; PCT / US2014 / 070413; PCT / US2015 / 36773; PCT / US2015 / 36759; PCT / US2015 / 36771; or PCT / IB2017 / 051367, all of which are incorporated herein by reference.

[0106] Accordingly, the nucleic acids of the present disclosure (e.g., DNA nucleic acids and RNA nucleic acids (such as mRNA nucleic acids)) can include standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

[0107] In some embodiments, the nucleic acids of the present disclosure (e.g., DNA nucleic acids and RNA nucleic acids (such as mRNA nucleic acids)) include various (two or more) different types of standard and / or modified nucleotides and nucleosides. In some embodiments, a particular region of the nucleic acid contains one, two, or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0108] In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms exhibit reduced degradation in the cells or organisms, respectively, compared to unmodified nucleic acids containing standard nucleotides and nucleosides.

[0109] In some embodiments, a modified RNA nucleic acid (e.g., a nucleic acid of modified mRNA) introduced into a cell or an organism may exhibit a reduction in immunogenicity (e.g., a reduction in innate response) in the cell or the organism, respectively, as compared to an unmodified nucleic acid containing standard nucleotides and nucleosides.

[0110] In some embodiments, a nucleic acid (e.g., an RNA nucleic acid (such as a nucleic acid of mRNA)) contains non-natural modified nucleotides introduced during or after the synthesis of the nucleic acid to achieve desired functions or properties. Modifications can be present on the internucleotide linkage, the purine or pyrimidine base, or the sugar. Modifications can be introduced by chemical synthesis or polymerase enzymes at the end of the strand or other parts in the strand. Any of the regions of the nucleic acid can be chemically modified.

[0111] The present disclosure provides modified nucleosides and nucleotides of nucleic acids (e.g., RNA nucleic acids (such as nucleic acids of mRNA)). "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleic acid base"). "Nucleotide" refers to a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method (e.g., chemically, enzymatically, or recombinantly, etc.) to include one or more modified nucleosides or non-natural nucleosides. A nucleic acid can include a region (s) of linked nucleosides. Such a region can have variable backbone linkages. The linkage is a standard phosphodiester linkage, and in that case, the nucleic acid will include a region of nucleotides.

[0112] Modified nucleotide base pairing encompasses not only standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and / or non-standard or modified bases, including modified nucleotides, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between a non-standard base and a standard base, or between two complementary non-standard base structures (such as those in a nucleic acid having at least one chemical modification). One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of bases / sugars or linkers can be incorporated into the nucleic acids of the present disclosure.

[0113] In some embodiments, modified nucleobases in a nucleic acid (such as an RNA nucleic acid (e.g., the nucleic acid of mRNA)) include 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (ψ). In some embodiments, modified nucleobases in a nucleic acid (such as an RNA nucleic acid (e.g., the nucleic acid of mRNA)) include 5-methoxymethyluridine, 5-methylthiouidine, 1-methoxymethylpseudouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, a polynucleotide includes a combination of at least two (e.g., two, three, four or more) of any of the foregoing modified nucleobases (including but not limited to chemical modifications).

[0114] In some embodiments, the RNA nucleic acids of the present disclosure include 1-methyl-pseudouridine (m1ψ) substitution at one or more or all uridine positions of the nucleic acid.

[0115] In some embodiments, the RNA nucleic acids of the present disclosure include 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions of the nucleic acid, and 5-methylcytidine substitutions at one or more or all cytidine positions of the nucleic acid.

[0116] In some embodiments, the RNA nucleic acids of the present disclosure include pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid.

[0117] In some embodiments, the RNA nucleic acids of the present disclosure include pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid, and 5-methylcytidine substitutions at one or more or all cytidine positions of the nucleic acid.

[0118] In some embodiments, the RNA nucleic acids of the present disclosure include uridine at one or more or all uridine positions of the nucleic acid.

[0119] In some embodiments, the nucleic acid (e.g., RNA nucleic acid such as the nucleic acid of mRNA) is uniformly modified for a particular modification (e.g., fully modified, modified throughout the entire sequence). For example, the nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the sequence of the mRNA are replaced by 1-methyl-pseudouridine. Similarly, the nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue (such as those described above).

[0120] The nucleic acids of the present disclosure can be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) can be uniformly modified in the nucleic acids of the present disclosure, or in its defined sequence region (e.g., in mRNA including or excluding the polyA tail). In some embodiments, all nucleotides X in the nucleic acids of the present disclosure (or in its sequence region) are modified nucleotides, where X can be any one of the nucleotides A, G, U, C, or any one of the combinations of A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C+U.

[0121] The nucleic acid can contain from about 1% to about 100%, or any percentage in between (e.g., 1% - 20%, 1% - 25%, 1% - 50%, 1% - 60%, 1% - 70%, 1% - 80%, 1% - 90%, 1% - 95%, 10% - 20%, 10% - 25%, 10% - 50%, 10% - 60%, 10% - 70%, 10% - 80%, 10% - 90%, 10% - 95%, 10% - 100%, 20% - 25%, 20% - 50%, 20% - 60%, 20% - 70%, 20% - 80%, 20% - 90%, 20% - 95%, 20% - 100%, 50% - 60%, 50% - 70%, 50% - 80%, 50% - 90%, 50% - 95%, 50% - 100%, 70% - 80%, 70% - 90%, 70% - 95%, 70% - 100%, 80% - 90%, 80% - 95%, 80% - 100%, 90% - 95%, 90% - 100%, and 95% - 100%) modified nucleotides (either in relation to the overall nucleotide content, or in relation to one or more types of nucleotides (i.e., any one or more of A, G, U or C)). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U or C.

[0122] The nucleic acid may contain nucleotides modified at a minimum of 1% and a maximum of 100%, or any percentage in between (at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides, etc.). For example, the nucleic acid may contain modified pyrimidines (modified uracil or cytosine). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced by modified uracil (e.g., 5-substituted uracil). The modified uracil may be replaced by a compound having a single unique structure, or may be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the nucleic acid is replaced by modified cytosine (e.g., 5-substituted cytosine). The modified cytosine may be replaced by a compound having a single unique structure, or may be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

[0123] Untranslated Region (UTR) The nucleic acids of the present disclosure can include one or more regions or portions that act or function as untranslated regions. When the nucleic acid is designed to encode at least one antigen of interest, the nucleic acid can include one or more of these untranslated regions (UTRs). The wild-type untranslated regions of the nucleic acid are transcribed but not translated. In mRNA, the 5’UTR starts at the transcription start site and continues to the start codon but does not include the start codon. However, the 3’UTR starts immediately following the stop codon and continues to the transcription termination signal. A number of evidences regarding the regulatory role played by UTRs with respect to nucleic acid molecules and translation stability are increasing. The regulatory features of UTRs can be incorporated into the polynucleotides of the present disclosure, particularly to promote the stability of the molecule. In cases where it is misdirected to an undesired organ site, specific features can be incorporated to ensure regulated downregulation of the transcript. A wide variety of 5’UTR sequences and 3’UTR sequences are known and available in the art.

[0124] The 5’UTR is the region of the mRNA that is immediately upstream (5’) of the start codon (the first codon of the mRNA transcript that is translated by the ribosome). The 5’UTR does not encode a protein (non-coding). Native 5’UTRs have features that play a role in translation initiation. They possess signatures such as the Kozak sequence (which is generally known to be involved in the process by which ribosomes initiate translation of many genes). The Kozak sequence has the consensus CCR(A / G)CCAUGG (SEQ ID NO: 121), where in the sequence, R is a purine (adenine or guanine) and is 3 bases upstream of the start codon (AUG) (followed by another “G”). The 5’UTR is also known to form secondary structures that are involved in elongation factor binding.

[0125] In some embodiments of the present disclosure, the 5’UTR is a heterologous UTR (i.e., a naturally-occurring UTR associated with a different ORF). In another embodiment, the 5’UTR is a synthetic UTR (i.e., not naturally occurring). Synthetic UTRs include those that are completely synthetic, in addition to UTRs that have been mutated to improve their properties (e.g., that increase gene expression). Exemplary 5’UTRs include a-globin or b-globin from Xenopus or human (8278063;9012219), human cytochrome b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and tobacco etch virus (US8278063, 9012219). The CMV immediate early 1 (IE1) gene (US20140206753, WO2013 / 185069), the sequence GGGAUCCUACC (SEQ ID NO: 122) (WO2014 / 144196) may also be used. In another embodiment, the 5’UTR of the TOP gene is the 5’UTR of a TOP gene lacking a 5’TOP motif (oligopyrimidine tract) (e.g., WO2015 / 101414, WO2015 / 101415, WO2015 / 062738, WO2015 / 024667, WO2015 / 024668). A 5’UTR element derived from the ribosomal protein large 32 (L32) gene (WO2015 / 101414, WO2015 / 101415, WO2015 / 062738), a 5’UTR element derived from the 5’UTR of the hydroxysteroid (17-β) dehydrogenase 4 gene (HSD17B4) (WO2015 / 024667), or a 5’UTR element derived from the 5’UTR of ATP5A1 (WO2015 / 024667) may be used. In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5’UTR.

[0126] In some embodiments, the 5’UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 104.

[0127] The 3’UTR is the region of mRNA immediately downstream (3’) from the stop codon (the codon of the mRNA transcript that is the signal for the end of translation). The 3’UTR does not code for protein (is non-coding). Natural or wild-type 3’UTRs are known to incorporate stretches of adenosine and uridine within them. These AU-rich signatures are particularly well seen in genes with high turnover rates. Based on their sequence features and functional properties, AU-rich elements (AREs) can be divided into three classes (Chen et al, 1995). Class I AREs contain multiple dispersed copies of the AUUUA motif within a U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs retain two or more overlapping UUAUUUA(U / A)(U / A) (SEQ ID NO: 123) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-a. Class III ARES are not well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this class. Most proteins that bind to AREs are known to destabilize the messenger, although members of the ELAV family (most notably HuR) have been reported to increase mRNA stability. HuR binds to AREs of all three classes. Manipulation of the specific binding sites for HuR within the 3’UTR of a nucleic acid molecule will lead to binding of HuR and thus stabilization of the message in vivo.

[0128] The stability of the nucleic acids (e.g., RNA) of the present disclosure can be regulated using introduction, removal, or modification of 3’UTR AU-rich elements (AREs). When manipulating specific nucleic acids, one or more copies of an ARE can be introduced to make the nucleic acids of the present disclosure more unstable, thereby suppressing translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability and thus increase the translation and production of the resulting protein. Transfection experiments are performed in appropriate cell lines using the nucleic acids of the present disclosure, and protein production can be assayed at various time points after transfection. For example, cells are transfected with different ARE-manipulating molecules, and the proteins produced at 6 hours, 12 hours, 24 hours, 48 hours, and 7 days after transfection are assayed using an ELISA kit for the appropriate protein.

[0129] The 3’UTR can be heterologous or synthetic. With respect to the 3’UTR, globin UTRs (including Xenopus β-globin UTR and human β-globin UTR) are known in the art (8278063, 9012219, US20110086907). By cloning two consecutive human β-globin 3’UTRs head-to-tail, a modified β-globin construct with enhanced stability in several cell types has been developed and is well-known in the art (US2012 / 0195936, WO2014 / 071963). In addition, the UTRs of a2-globin, a1-globin and their mutations are also known in the art (WO2015 / 101415, WO2015 / 024667). Other 3’UTRs described in the mRNA constructs in the non-patent literature include CYBA (Ferizi et al., 2015) and albumin (Thess et al., 2015). Other exemplary 3’UTRs include those of bovine or human growth hormone (wild-type or modified) (WO2013 / 185069, US20140206753, WO2014 / 152774), rabbit β-globin and hepatitis B virus (HBV), and the α-globin 3’UTR and viral VEEV 3’UTR sequences are also known. In some embodiments, the sequence UUUGAAUU (WO2014 / 144196) is used. In some embodiments, the 3’UTRs of human and mouse ribosomal proteins are used. Other examples include rps9 3’UTR (WO2015 / 101414), FIG4 (WO2015 / 101415) and human albumin 7 (WO2015 / 101415).

[0130] In some embodiments, the 3’UTR of the present disclosure comprises a sequence selected from SEQ ID NO:3 and SEQ ID NO:106.

[0131] One of ordinary skill in the art will understand that a heterologous or synthetic 5’UTR can be used with any desired 3’UTR sequence. For example, a heterologous 5’UTR can be used with a synthetic 3’UTR having a heterologous 3’UTR.

[0132] Non-UTR sequences can also be used as regions or sub-regions within a nucleic acid. For example, the sequence of an intron or a portion of an intron sequence can be incorporated into the regions of the nucleic acids of the present disclosure. Incorporation of intronic sequences can also increase protein production in addition to the nucleic acid level.

[0133] Combinations of features can be included in adjacent regions or contained within other features. For example, an ORF can be flanked by a 5'UTR that can contain a strong Kozak translation initiation signal and / or a 3'UTR that can contain an oligo(dT) sequence for template addition of a polyA tail. The 5'UTR can include a first polynucleotide fragment as well as a second polynucleotide fragment from the same gene and / or a different gene (such as the 5'UTRs described in U.S. Patent Application Publication No. 20100293625 and PCT / US2014 / 069155, which are hereby incorporated by reference in their entirety).

[0134] It should be understood that any UTR from any gene can be incorporated into a nucleic acid region. Further, multiple wild-type UTRs of any known gene can be utilized. It is also within the scope of the present disclosure to provide artificial UTRs that are not variants of the wild-type region. These UTRs or portions thereof can be placed in the same orientation as in the transcript in which they are selected, or the orientation or location can be altered. Thus, a 5'UTR or 3'UTR can be inverted, shortened, or extended along with one or more other 5'UTRs or 3'UTRs. As used herein, the term "altered," when related to a UTR sequence, means that the UTR is changed in some way relative to a reference sequence. For example, a 3'UTR or 5'UTR can be altered compared to a wild-type or native UTR by a change in orientation or location as taught above, or by the incorporation of additional nucleotides, nucleotide deletions, nucleotide swapping, or translocation. Any of these changes that produce an "altered" UTR (regardless of 3' or 5') include variant UTRs.

[0135] In some embodiments, double, triple, or quadruple UTRs (such as 5'UTRs or 3'UTRs) can be used. As used herein, a "double" UTR is one in which two copies of the same UTR are encoded either continuously or substantially continuously. For example, a double beta-globin 3'UTR can be used as described in U.S. Patent Publication 20100129877, the content of which is incorporated herein by reference in its entirety.

[0136] It is also within the scope of the present disclosure to have patterned UTRs. As used herein, a "patterned UTR" is a UTR that reflects a repeating or alternating pattern (such as ABABAB or AABBAABBAABB or ABCABCABC, or variants thereof that repeat one, two, or three or more times). In these patterns, each letter (A, B, or C) represents a different UTR at the nucleotide level.

[0137] In some embodiments, the adjacent regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, the polypeptide of interest may belong to a family of proteins that are expressed in a particular cell, tissue or at a certain time during development. The UTRs from any of these genes can be swapped with other UTRs of the same or different families of proteins to generate novel polynucleotides. As used herein, "family of proteins" is used in the broadest sense to refer to a group of two or more polypeptides of interest that share at least one function, structure, feature, localization, origin, or expression pattern.

[0138] The untranslated region may also include a translation enhancer element (TEE). By way of non-limiting example, TEEs may include those described in U.S. Application No. 20090226470, which is incorporated herein by reference in its entirety, and those known in the art.

[0139] In Vitro Transcription of RNA The cDNA encoding the polynucleotide described herein can be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is known in the art and is described in International Publication WO / 2014 / 152027, which is incorporated herein by reference in its entirety.

[0140] In some embodiments, the RNA transcript is generated using a non-amplified linearized DNA template in an in vitro transcription reaction to produce the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of an RNA polynucleotide and is not limited to, for example, EBV RNA (e.g., EBV mRNA). In some embodiments, cells (e.g., bacterial cells, e.g., E. coli, e.g., DH-1 cells) are transfected with a plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA, which is then isolated and purified. In some embodiments, the DNA template contains an RNA polymerase promoter (e.g., T7 promoter) located at the 5' and operably linked to the gene of interest.

[0141] In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a polyA tail. The specific nucleic acid sequence composition and length of the in vitro transcription template will depend on the mRNA encoded by the template.

[0142] The "5' untranslated region" (UTR) refers to the region of the mRNA that does not encode a polypeptide and is immediately upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript that is translated by the ribosome). When an RNA transcript is being generated, the 5' UTR may contain a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in the vaccines of the present disclosure.

[0143] The "3' untranslated region" (UTR) refers to the region of the mRNA that does not encode a polypeptide and is immediately downstream (i.e., 3') of the stop codon (i.e., the codon of the mRNA transcript that is a signal for the end of translation).

[0144] An "open reading frame" is a continuous stretch of DNA that starts with a start codon (e.g., methionine (ATG)), ends with a stop codon (e.g., TAA, TAG, or TGA), and encodes a polypeptide.

[0145] A "polyA tail" is a region of mRNA that is downstream (e.g., immediately downstream (i.e., 3')) from the 3'UTR and contains a plurality of consecutive adenosine monophosphates. The polyA tail can contain 10 to 300 adenosine monophosphates. For example, the polyA tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, the polyA tail contains 50 to 250 adenosine monophosphates. In a suitable biological setting (e.g., in cells in vivo), the poly(A) tail functions, for example, to protect the mRNA from degradation by enzymes in the cytoplasm and to assist in transcription termination and / or export of the mRNA from the nucleus and translation.

[0146] In some embodiments, the nucleic acid comprises 200 to 3,000 nucleotides. For example, the nucleic acid can comprise 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

[0147] An in vitro transcription system typically includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase.

[0148] NTPs can be manufactured in-house, selected from a supplier, or synthesized as described herein. NTPs can be selected from, but not limited to, those described herein and include natural and non-natural (modified) NTPs.

[0149] Any number of RNA polymerases or variants can be used in the methods of the disclosure. The polymerase can be selected from, but not limited to, phage RNA polymerases (such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase), and / or mutant polymerases (such as polymerases capable of incorporating modified nucleic acids and / or modified nucleotides, including chemically modified nucleic acids and / or nucleotides). Some embodiments exclude the use of DNase.

[0150] In some embodiments, the RNA transcript is capped via enzymatic capping. In some embodiments, the RNA contains a 5' end cap (such as 7mG(5')ppp(5')NlmpNp).

[0151] Chemical synthesis Solid-phase chemical synthesis. The nucleic acids of the disclosure can be produced in whole or in part using solid-phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method where the molecule is immobilized on a solid support and synthesized stepwise in a solution of reactants. Solid-phase synthesis is useful for the site-specific introduction of chemical modifications in nucleic acid sequences.

[0152] Liquid-phase chemical synthesis. The synthesis of the nucleic acids of the disclosure can be carried out in the liquid phase by the sequential addition of monomer building blocks.

[0153] Combinations of synthesis methods. Each of the synthesis methods discussed above has its own advantages and limitations. In order to overcome these limitations, attempts have been made to combine these methods. Such combinations of methods are within the scope of the present disclosure. The use of solid-phase chemical synthesis or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient approach to generate nucleic acid chains of lengths that cannot be obtained by chemical synthesis alone.

[0154] Ligation of regions or sub-regions of nucleic acids Assembly of nucleic acids by ligase can also be used. DNA ligase or RNA ligase promotes intermolecular ligation of the 5' and 3' ends of polynucleotide chains through the formation of phosphodiester bonds. Nucleic acids such as chimeric polynucleotides and / or circular nucleic acids can be prepared by ligation of one or more regions or sub-regions. DNA fragments can be joined by a ligase-catalyzed reaction to generate recombinant DNA with different functions. Two oligodeoxynucleotides (one with a 5' phosphoryl group and another with a free 3' hydroxyl group) are provided as substrates for DNA ligase.

[0155] Purification The purification of nucleic acids described herein may include, but is not limited to, nucleic acid clean-up, quality assurance, and quality control. Clean-up can be performed by methods known in the art (such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark), etc.) or HPLC-based purification methods (such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC)). The term "purified", when used in relation to a nucleic acid ("purified nucleic acid", etc.), refers to that which has been separated from at least one contaminant. A "contaminant" is any substance that renders another substance inappropriate, impure, or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) exists in a form or configuration different from that in which it is found in nature, or in a form or configuration different from that which existed before any treatment or purification method was performed on it.

[0156] Quality assurance and / or quality control checks can be performed using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

[0157] In some embodiments, nucleic acids can be sequenced by methods such as, but not limited to, reverse transcriptase-PCR.

[0158] Quantification In some embodiments, when derived from one or more body fluids, the nucleic acids of the present invention can be quantified in exosomes. Body fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and ascitic fluid, pericardial fluid, lymph, chyle, chyle, bile, interstitial fluid, menstruation, pus, sebum, vomit, vaginal secretion, mucosal secretion, fecal water, pancreatic juice, nasal wash fluid, bronchopulmonary aspirate, blastocyst cavity body fluid, and umbilical cord blood. Alternatively, exosomes can be recovered from organs selected from the group consisting of the lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, large intestine, breast, prostate, brain, esophagus, liver, and placenta.

[0159] The assay can be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof, while exosomes can be isolated using immunohistochemistry (such as enzyme-linked immunosorbent assay (ELISA) methods). Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or combinations thereof.

[0160] These methods give the investigator the ability to monitor in real time the levels of residual or delivered nucleic acids. This is possible because the nucleic acids of the present disclosure differ from their endogenous forms in some embodiments due to structural or chemical modifications.

[0161] In some embodiments, the nucleic acid can be quantified using methods such as, but not limited to, ultraviolet-visible spectroscopy (UV / Vis). Non-limiting examples of UV / Vis spectrometers are NANODROP® spectrometers (ThermoFisher, Waltham, MA). The quantified nucleic acid can be analyzed to determine if the nucleic acid is of appropriate size and to check that no degradation of the nucleic acid has occurred. Degradation of the nucleic acid can be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC-based purification methods (such as strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC)), liquid chromatography mass spectrometry (LCMS), capillary electrophoresis (CE), and capillary gel electrophoresis (CGE).

[0162] Pharmaceutical formulation Compositions (e.g., pharmaceutical compositions), methods, kits, and reagents are provided herein for the prevention or treatment of EBV in, for example, humans and other mammals. An EBV RNA (e.g., mRNA) vaccine can be used as a therapeutic or prophylactic agent. They can be used in a medicament to prevent and / or treat an infectious disease.

[0163] In some embodiments, an EBV vaccine containing an RNA polynucleotide as described herein can be administered to a subject (e.g., a mammalian subject such as a human subject), and the RNA polynucleotide is translated in vivo to produce an antigenic polypeptide (antigen).

[0164] The "effective amount" of an EBV vaccine is at least partially based on the target tissue, target cell type, means of administration, physical characteristics of the RNA (such as length, nucleotide composition, and / or degree of modified nucleosides), other components of the vaccine, and other determining factors (such as the age, weight, height, sex, and general health of the subject). Typically, the effective amount of an EBV vaccine provides an induced or boosted immune response as a function of antigen production in the cells of the subject. In some embodiments, the effective amount of an EBV RNA vaccine containing an RNA polynucleotide having at least one chemical modification is more efficient than a composition containing the corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. The increase in antigen production can be demonstrated by an increase in cell transfection (percentage of cells transfected with the RNA vaccine), an increase in protein translation and / or expression from the polynucleotide, a decrease in nucleic acid degradation (as demonstrated, for example, by an increase in the duration of protein translation from the modified polynucleotide), or a change in the antigen-specific immune response of the host cell.

[0165] The term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier that makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable carrier" does not cause any undesirable physiological effects after administration to or on a subject. The carrier in a pharmaceutical composition must also be "acceptable" in the sense that it is compatible with the active ingredient and capable of stabilizing the active ingredient. One or more solubilizing agents can be utilized as pharmaceutical carriers for the delivery of the active agent. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents that achieve a composition usable as a dosage form. Other examples of carriers include colloidal silicon dioxide, magnesium stearate, cellulose, and sodium lauryl sulfate. In addition to additional suitable pharmaceutical carriers and diluents, pharmaceutical necessities for their use are described in Remington’s Pharmaceutical Sciences.

[0166] In some embodiments, RNA vaccines (including polynucleotides and their encoded polypeptides) according to the present disclosure can be used for the treatment or prevention of EBV. The EBV RNA vaccine can be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals, or during the early stages of infection during the incubation phase, or during active infection after the onset of symptoms. In some embodiments, the amount of the RNA vaccine of the present disclosure provided to a cell, tissue, or subject can be an amount effective for immunoprophylaxis.

[0167] An EBV RNA (e.g., mRNA) vaccine can be administered together with other prophylactic or therapeutic compounds. As non-limiting examples, the prophylactic or therapeutic compound can be an adjuvant or a booster. As used herein, when referring to a prophylactic composition (such as a vaccine), the term "booster" refers to an additional administration of a prophylactic (vaccine) composition. A booster (or booster vaccine) can be given after an initial administration of the prophylactic composition. The time between the first administration of the prophylactic composition and the booster can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or 99 years or more, but is not limited thereto. In an exemplary embodiment, the time between the first administration of the prophylactic composition and the booster can be 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year, but is not limited thereto.

[0168] In some embodiments, the EBV RNA vaccine can be administered intramuscularly, intranasally, or intradermally, similar to the administration of inactivated vaccines known in the art.

[0169] EBV RNA vaccines can be utilized in various settings depending on the incidence of infection, or the degree or level of unmet medical need. By way of non-limiting example, RNA vaccines can be used to treat and / or prevent various infectious diseases. RNA vaccines have excellent properties in that they produce much higher antibody titers, better neutralizing immunity, a more persistent immune response, and / or produce a response more quickly than commercially available vaccines.

[0170] Provided herein are pharmaceutical compositions comprising an EBV RNA vaccine and an RNA vaccine composition and / or complex, optionally in combination with one or more pharmaceutically acceptable excipients.

[0171] An EBV RNA (e.g., mRNA) vaccine can be formulated or administered alone or in combination with one or more other components. For example, an EBV RNA vaccine (vaccine composition) can include other components (including but not limited to adjuvants).

[0172] In some embodiments, the EBV RNA vaccines are adjuvant-free (they are adjuvant-inactive).

[0173] EBV RNA (e.g., mRNA) vaccines can be formulated or administered in combination with one or more pharmaceutically acceptable excipients. In some embodiments, the vaccine composition comprises at least one additional active substance (e.g., a therapeutically active substance, a prophylactically active substance, or a combination of both, etc.). The vaccine composition can be sterilized, can be pyrogen-free, or can be both sterilized and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents (such as vaccine compositions) can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (which is hereby incorporated by reference in its entirety).

[0174] In some embodiments, the EBV RNA vaccine is administered to a human, a human patient, or a subject. For the purposes of the present disclosure, the phrase "active ingredient" generally refers to an RNA vaccine or the polynucleotide contained therein (e.g., an RNA polynucleotide encoding an antigen (e.g., an mRNA polynucleotide)).

[0175] The formulations of the vaccine compositions described herein can be prepared by any method known in the art of pharmacology or developed in the future. Generally, such preparation methods include combining the active ingredient (e.g., the polynucleotide of mRNA) with excipients and / or one or more other auxiliary components, and then, if necessary and / or desired, dividing, shaping, and / or packaging the product into the desired single-dose units or multi-dose units.

[0176] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional components in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, as well as further depending on the route by which the composition is administered. By way of example, the composition may contain the active ingredient at between 0.1% and 100% (e.g., between 0.5 and 50%, between 1 and 30%, between 5 and 80%, at least 80%) (w / w).

[0177] In some embodiments, the EBV RNA vaccine is formulated using one or more excipients to (1) increase stability; (2) increase cell transfection; (3) enable sustained or delayed release (e.g., from a depot formulation); (4) alter biodistribution (e.g., target specific tissues or cell types); (5) increase translation of the encoded protein in vivo; and / or (6) alter the release profile of the encoded protein (antigen) in vivo. In addition to conventional excipients (any and all solvents, dispersion solvents, diluents or other liquid vehicles, agents for assisting dispersion or suspension, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, etc.), excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected by the EBV RNA vaccine (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0178] Lipid nanoparticles (LNP) In some embodiments, the EBV RNA (e.g., mRNA) vaccines of the present disclosure are formulated in lipid nanoparticles (LNPs). Lipid nanoparticles typically include ionizable cationic lipids, non-cationic lipids, sterols, and PEG lipid components, together with the nucleic acid cargo of interest. The lipid nanoparticles of the present disclosure can be generated using components, compositions, and methods as generally known in the art, see, for example, PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129; PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 (all of which are incorporated herein by reference in their entirety).

[0179] The vaccines of the present disclosure are typically formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles include at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)-modified lipid.

[0180] In some embodiments, the lipid nanoparticles include ionizable cationic lipids in a molar ratio of 20-60%. For example, the lipid nanoparticles can include ionizable cationic lipids in a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50% or 50-60%. In some embodiments, the lipid nanoparticles include ionizable cationic lipids in a molar ratio of 20%, 30%, 40%, 50 or 60%.

[0181] In some embodiments, the lipid nanoparticles contain 5-25% non-cationic lipid by molar ratio. For example, the lipid nanoparticles may contain 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20% or 20-25% non-cationic lipid by molar ratio. In some embodiments, the lipid nanoparticles contain 5%, 10%, 15%, 20% or 25% non-cationic lipid by molar ratio.

[0182] In some embodiments, the lipid nanoparticles contain 25-55% sterol by molar ratio. For example, the lipid nanoparticles may contain 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50% or 50-55% sterol by molar ratio. In some embodiments, the lipid nanoparticles contain 25%, 30%, 35%, 40%, 45%, 50% or 55% sterol by molar ratio.

[0183] In some embodiments, the lipid nanoparticles contain 0.5-15% PEG-modified lipid by molar ratio. For example, the lipid nanoparticles may contain 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10% or 10-15% by molar ratio. In some embodiments, the lipid nanoparticles contain 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% PEG-modified lipid by molar ratio.

[0184] In some embodiments, the lipid nanoparticles contain 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid by molar ratio.

[0185] In some embodiments, the ionizable cationic lipid of the present disclosure is a compound of formula (I):

Chemical formula

[0186] In some embodiments, a subset of the compounds of formula (I) includes those wherein, R 4 is -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR or -CQ(R) 2 and when that is the case, then (i) when n is 1, 2, 3, 4 or 5, Q is not -N(R) 2 or, (ii) when n is 1 or 2, Q is not a 5-, 6- or 7-membered heterocycloalkyl.

[0187] In some embodiments, another subset of the compounds of formula (I) includes those, or salts or isomers thereof, wherein, R 1 is C 5-30 alkyl, C 5-20selected from the group consisting of alkenyl, -R*YR”, -YR” and -R”M’R’; R 2 and R 3 is H, C 1-14 alkyl, C 2-14 alkenyl, -R*YR”, -YR” and -R*OR” independently selected from the group consisting of, or R 2 and R 3 together with the atom to which they are attached form a heterocyclic or carbocyclic ring; R 4 is C 3-6 carbocyclic ring, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R) 2 and unsubstituted C 1-6 alkyl selected from the group consisting of, wherein Q is C 3-6 carbocyclic ring, N, O and S having one or more heteroatoms selected from 5- to 14-membered heteroaryl, -OR, -O(CH 2 ) n N(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 , -CRN(R) 2 C(O)OR, -N(R)R 8 , -O(CH 2 ) n OR, -N(R)C(=NR 9 )N(R) 2 , -N(R)C(=CHR 9 )N(R) 2 , -OC(O)N(R) 2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2 R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2, -N(OR)C(S)N(R) 2 , -N(OR)C(=NR 9 )N(R) 2 , -N(OR)C(=CHR 9 )N(R) 2 , -C(=NR 9 )N(R) 2 , -C(=NR 9 )R, -C(O)N(R)OR, and oxo(=O), OH, amino, monoalkylamino or dialkylamino and C 1-3 selected from 5- to 14-membered heterocycloalkyl having one or more heteroatoms selected from N, O and S, substituted by one or more substituents selected from C each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl and H; each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 -, -S-S-, aryl group and heteroaryl group; R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl and H; R 8 is selected from the group consisting of C 3-6 carbocyclic ring and heterocyclic ring; R 9 is H, CN, NO 2 , C 1-6 alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2-6 alkenyl, C3-6 selected from the group consisting of carbon rings and hetero rings; each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H; each R' is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, -R*YR", -YR", and H; each R" is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl; each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl; each Y is independently C 3-6 a carbon ring; each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0188] In some embodiments, another subset of the compounds of formula (I) includes those as follows, or salts or isomers thereof, wherein R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, -R*YR", -YR", and -R"M'R'; R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 together with the atom to which they are attached form a hetero ring or a carbon ring; R 4 is C 3-6 a carbon ring, -(CH 2 ) n Q, -(CH 2 ) nCHQR, -CHQR, -CQ(R) 2 and unsubstituted C 1-6 selected from the group consisting of alkyl, wherein Q is C 3-6 a 5- to 14-membered heterocyclic ring having one or more heteroatoms selected from carbocyclic ring, N, O and S, -OR, -O(CH 2 ) n N(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 , -CRN(R) 2 C(O)OR, -N(R)R 8 , -O(CH 2 ) n OR, -N(R)C(=NR 9 )N(R) 2 , -N(R)C(=CHR 9 )N(R) 2 , -OC(O)N(R) 2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2 R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2 , -N(OR)C(S)N(R) 2 , -N(OR)C(=NR 9 )N(R) 2 , -N(OR)C(=CHR 9 )N(R) 2 , -C(=NR 9 )R, -C(O)N(R)OR and -C(=NR 9 )N(R) 2 selected from, and each n is independently selected from 1, 2, 3, 4 and 5; Q is a 5- to 14-membered heterocyclic ring, and (i) R 4 is (CH 2 ) n Q (wherein n is 1 or 2), or (ii) R 4 is (CH 2 )n is CHQR (where n is 1), or (iii) R 4 is CHQR and CQ(R) 2 when that is the case, then Q is either a 5- to 14-membered heteroaryl or an 8- to 14-membered heterocycloalkyl; each R 5 is C 1-3 alkyl, C 2-3 alkenyl and H, independently selected from the group consisting of; each R 6 is C 1-3 alkyl, C 2-3 alkenyl and H, independently selected from the group consisting of; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O) 2 -, -S-S-, aryl groups and heteroaryl groups; R 7 is C 1-3 alkyl, C 2-3 alkenyl and H, selected from the group consisting of; R 8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R 9 is H, CN, NO 2 , C 1-6 alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; each R is C 1-3 alkyl, C 2-3 alkenyl and H, independently selected from the group consisting of; each R’ is C 1-18 alkyl, C 2-18 alkenyl, -R*YR”, -YR” and H, independently selected from the group consisting of; each R” is C3-14 Alkyl and C 3-14 is independently selected from the group consisting of alkenyl; each R* is C 1-12 alkyl and C 2-12 is independently selected from the group consisting of alkenyl; each Y is independently C 3-6 is a carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0189] In some embodiments, another subset of the compounds of formula (I) includes those as follows, or salts or isomers thereof, wherein R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R 2 and R 3 together with the atom to which they are attached form a heterocycle or a carbocycle; R 4 is selected from the group consisting of C 3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R) 2 and unsubstituted C 1-6 alkyl, wherein Q is C 3-6 carbocycle, 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH 2 ) n N(R) 2 -, -C(O)OR, -OC(O)R, -CX 3 -, -CX2 H, -CXH 2 , -CN, -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 , -CRN(R) 2 C(O)OR, -N(R)R 8 , -O(CH 2 ) n OR, -N(R)C(=NR 9 )N(R) 2 , -N(R)C(=CHR 9 )N(R) 2 , -OC(O)N(R) 2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2 R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2 , -N(OR)C(S)N(R) 2 , -N(OR)C(=NR 9 )N(R) 2 , -N(OR)C(=CHR 9 )N(R) 2 , -C(=NR 9 )R, -C(O)N(R)OR and -C(=NR 9 )N(R) 2 selected from, and each n is independently selected from 1, 2, 3, 4 and 5; each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl and H; each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 (-), -S-S-, aryl groups and heteroaryl groups; R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl and H; R 8 is selected from the group consisting of C 3-6 carbocyclic and heterocyclic; R 9 is H, CN, NO 2 , C 1-6 alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocyclic and heterocyclic; Each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl and H; Each R' is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, -R*YR", -YR" and H; Each R" is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl; Each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl; Each Y is independently C 3-6 carbocyclic; Each X is independently selected from the group consisting of F, Cl, Br and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13.

[0190] In some embodiments, another subset of the compounds of formula (I) includes those as follows, or salts or isomers thereof, wherein R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, -R*YR", -YR" and -R"M'R'; R2 and R 3 is H, C 2-14 alkyl, C 2-14 alkenyl, -R*YR”, -YR” and -R*OR” independently selected from the group consisting of, or R 2 and R 3 together with the atom to which they are attached form a heterocyclic or carbocyclic ring; R 4 is -(CH 2 ) n Q or -(CH 2 ) n CHQR (where Q is -N(R) 2 and n is selected from 3, 4 and 5); each R 5 is C 1-3 alkyl, C 2-3 alkenyl and H independently selected from the group consisting of; each R 6 is C 1-3 alkyl, C 2-3 alkenyl and H independently selected from the group consisting of; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O) 2 -, -S-S-, aryl group and heteroaryl group; R 7 is C 1-3 alkyl, C 2-3 alkenyl and H selected from the group consisting of; each R is C 1-3 alkyl, C 2-3 alkenyl and H independently selected from the group consisting of; each R’ is C 1-18 alkyl, C 2-18 alkenyl, -R*YR”, -YR” and H independently selected from the group consisting of; each R” is C 3-14 alkyl and C 3-14 alkenyl independently selected from the group consisting of; Each R* is C 1-12 alkyl and C 1-12 independently selected from the group consisting of alkenyl; each Y is independently C 3-6 a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0191] In some embodiments, another subset of the compounds of formula (I) includes those as follows, or salts or isomers thereof, wherein R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R 2 and R 3 are independently selected from the group consisting of C 1-14 alkyl, C 2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R 2 and R 3 together with the atom to which they are attached form a heterocyclic or carbocyclic ring; R 4 is -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, and -CQ(R) 2 (wherein Q is -N(R) 2 and n is selected from 1, 2, 3, 4, and 5) and is selected from the group consisting of; each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H; each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O) 2 -, -S-S-, aryl groups, and heteroaryl groups; R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H; each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl; each R* is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl; each Y is independently a C 3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0192] In some embodiments, a subset of the compounds of formula (I) are those of formula (IA):

Chemical formula

[0193] In some embodiments, a subset of the compounds of formula (I) are those of formula (II):

Chemical formula

[0194] In some embodiments, a subset of the compounds of formula (I) are those of formula (IIa), (IIb), (IIc) or (IIe):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0195] In some embodiments, a subset of the compounds of formula (I) are those of formula (IId):

Chemical formula

[0196] In some embodiments, the ionizable cationic lipid of the present disclosure is a compound having the following structure: [Chemical formula] including.

[0197] In some embodiments, the ionizable cationic lipid of the present disclosure is a compound having the following structure: [Chemical formula] including.

[0198] In some embodiments, the non-cationic lipids of the present disclosure include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2 cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0199] In some embodiments, the PEGylated lipids of the present disclosure include PEGylated phosphatidylethanolamine, PEGylated phosphatidic acid, PEGylated ceramide, PEGylated dialkylamine, PEGylated diacylglycerol, PEGylated dialkylglycerol, and mixtures thereof. In some embodiments, the PEGylated lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and / or PEG-DPG.

[0200] In some embodiments, the sterols of the present disclosure include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and mixtures thereof.

[0201] In some embodiments, the LNP of the present disclosure includes the ionizable cationic lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG-DMG.

[0202] In some embodiments, the LNP of the present disclosure includes an N:P ratio of about 2:1 to about 30:1.

[0203] In some embodiments, the LNP of the present disclosure includes an N:P ratio of about 6:1.

[0204] In some embodiments, the LNP of the present disclosure includes an N:P ratio of about 3:1.

[0205] In some embodiments, the LNP of the present disclosure includes a wt / wt ratio of ionizable cationic lipid component to RNA of about 10:1 to about 100:1.

[0206] In some embodiments, the LNP of the present disclosure includes a wt / wt ratio of ionizable cationic lipid component to RNA of about 20:1.

[0207] In some embodiments, the LNPs of the present disclosure comprise a wt / wt ratio of ionizable cationic lipid component to RNA of about 10:1.

[0208] In some embodiments, the LNPs of the present disclosure have an average diameter of about 50 nm to about 150 nm.

[0209] In some embodiments, the LNPs of the present disclosure have an average diameter of about 70 nm to about 120 nm.

[0210] Multivalent vaccine The EBV vaccines provided herein may comprise one RNA (e.g., mRNA) or multiple RNAs encoding two or more antigens of the same or different EBV species. In some embodiments, the EBV vaccine comprises one RNA or multiple RNAs encoding two or more antigens selected from the gp350 antigen, gH antigen, gL antigen, gB antigen, gp42 antigen, LMP1 antigen, LMP2 antigen, EBNA1 antigen, and EBNA3 antigen. In some embodiments, the RNA (at least one RNA) of the EBV vaccine may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more antigens.

[0211] In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen and a gH antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen and a gL antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen and a gB antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen and a gp42 antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen and an LMP (e.g., LMP1 and / or LMP2) antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen and an EBNA (e.g., EBNA1 and / or EBNA3) antigen.

[0212] In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gH antigen, and a gL antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gH antigen, and a gB antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gH antigen, and a gp42 antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gH antigen, and an LMP (e.g., LMP1 and / or LMP2) antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gH antigen, and an EBNA (e.g., EBNA1 and / or EBNA3) antigen.

[0213] In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gL antigen, and a gB antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gL antigen, and a gp42 antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gL antigen, and an LMP (e.g., LMP1 and / or LMP2) antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gL antigen, and an EBNA (e.g., EBNA1 and / or EBNA3) antigen.

[0214] In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gB antigen, and a gp42 antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gB antigen, and an LMP (e.g., LMP1 and / or LMP2) antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gB antigen, and an EBNA (e.g., EBNA1 and / or EBNA3) antigen.

[0215] In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gp42 antigen, and an LMP (e.g., LMP1 and / or LMP2) antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, a gp42 antigen, and an EBNA (e.g., EBNA1 and / or EBNA3) antigen.

[0216] In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp350 antigen, an LMP (e.g., LMP1 and / or LMP2) antigen, and an EBNA (e.g., EBNA1 and / or EBNA3) antigen.

[0217] In some embodiments, the EBV vaccine comprises at least one RNA encoding a gH antigen and a gL antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gH antigen and a gB antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gH antigen and a gp42 antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gH antigen and an LMP (e.g., LMP1 and / or LMP2) antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gH antigen and an EBNA (e.g., EBNA1 and / or EBNA3) antigen.

[0218] In some embodiments, the EBV vaccine comprises at least one RNA encoding a gL antigen and a gB antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gL antigen and a gp42 antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gL antigen and an LMP (e.g., LMP1 and / or LMP2) antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gL antigen and an EBNA (e.g., EBNA1 and / or EBNA3) antigen.

[0219] In some embodiments, the EBV vaccine comprises at least one RNA encoding a gB antigen and a gp42 antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gB antigen and an LMP (e.g., LMP1 and / or LMP2) antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gB antigen and an EBNA (e.g., EBNA1 and / or EBNA3) antigen.

[0220] In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp42 antigen and an LMP (such as LMP1 and / or LMP2) antigen. In some embodiments, the EBV vaccine comprises at least one RNA encoding a gp42 antigen and an EBNA (such as EBNA1 and / or EBNA3) antigen.

[0221] In some embodiments, the EBV vaccine comprises at least one RNA encoding an LMP (such as LMP1 and / or LMP2) antigen and an EBNA (such as EBNA1 and / or EBNA3) antigen.

[0222] In some embodiments, two or more different RNAs (such as mRNAs) encoding antigens can be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNAs encoding antigens can be formulated in separate lipid nanoparticles (each RNA is formulated in a single lipid nanoparticle). The lipid nanoparticles can then be combined and administered as a single vaccine composition (such as comprising multiple RNAs encoding multiple antigens) or administered separately.

[0223] Combination vaccine The EBV vaccines provided herein can comprise one or more RNAs encoding two or more antigens of the same or different EBV strains. Also provided herein are combination vaccines comprising one or more EBV antigen(s) and RNA encoding one or more antigen(s) of different organisms (such as bacterial organisms and / or viral organisms). Thus, the vaccines of the disclosure can be combination vaccines that target one or more antigens of the same strain / species or one or more antigens of different strains / species (such as antigens that induce immunity against organisms found in the same geographic region where the risk of EBV infection is high or organisms to which an individual is likely to be exposed when exposed to EBV).

[0224] Dosage / Administration Compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of EBV in humans and other mammals are provided herein. The EBV RNA vaccine can be used as a therapeutic or prophylactic agent. In some embodiments, the RNA vaccine of the disclosure is used to provide prophylactic protection from EBV. In some embodiments, the RNA vaccine of the disclosure is used to treat EBV infection. In some embodiments, the EBV vaccine of the disclosure is used during the priming of immune effector cells to, for example, activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into the subject.

[0225] The subject can be any mammal (including non-human primates and human subjects). Typically, the subject is a human subject.

[0226] In some embodiments, the EBV vaccine is administered to a subject (e.g., a mammalian subject such as a human subject) in an amount effective to induce an antigen-specific immune response. The RNA encoding the EBV antigen is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject.

[0227] Prophylactic protection from EBV can be achieved following administration of the EBV RNA vaccine of the disclosure. The vaccine can be administered once, twice, three times, four times, or more, although administration of the vaccine once (optionally followed by a single booster) will likely be sufficient. Although less desirable, it is possible to administer the vaccine to an infected individual to achieve a therapeutic response. The dosage may need to be adjusted accordingly.

[0228] Methods of eliciting an immune response against EBV are provided in aspects of the present disclosure. The methods include administering to a subject an EBV RNA vaccine comprising at least one RNA (e.g., mRNA) having an open reading frame encoding at least one EBV antigen, thereby inducing an immune response specific for the EBV antigen in the subject, wherein the titer of the antigen-specific antibody in the subject is increased after vaccination as compared to the titer of the antigen-specific antibody in a subject vaccinated with a conventional vaccine against EBV at a prophylactically effective dose. "Antigen-specific antibody" is a serum antibody that specifically binds to an antigen.

[0229] A prophylactically effective dose is an effective dose that prevents viral infection at clinically acceptable levels. In some embodiments, the effective dose is the dose listed for the vaccine in the package insert. A conventional vaccine, as used herein, refers to a vaccine other than the mRNA vaccines of the present disclosure. For example, conventional vaccines include, but are not limited to, live microbial vaccines, killed microbial vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, virus-like particle (VLP) vaccines, and the like. In an exemplary embodiment, a conventional vaccine is a vaccine that has been regulatory approved and / or registered by a national drug regulatory agency (e.g., the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA)).

[0230] In some embodiments, the titer of the anti-antigen antibody in the subject is increased by 1 log to 10 log after vaccination compared to the titer of the anti-antigen antibody in a subject vaccinated with a conventional vaccine against EBV at a prophylactically effective dose or in a non-vaccinated subject. In some embodiments, the titer of the anti-antigen antibody in the subject is increased by 1 log, 2 log, 3 log, 4 log, 5 log or 10 log after vaccination compared to the titer of the anti-antigen antibody in a subject vaccinated with a conventional vaccine against EBV at a prophylactically effective dose or in a non-vaccinated subject.

[0231] Methods of eliciting an immune response in a subject against EBV are provided in other aspects of the disclosure. The method includes administering to the subject an EBV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one EBV antigen, thereby inducing an immune response specific for the EBV antigen in the subject, wherein the immune response in the subject is equivalent to the immune response in a subject vaccinated with a conventional vaccine against EBV at a dosing level that is 2-fold to 100-fold higher than the RNA vaccine.

[0232] In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a conventional vaccine at a dosage level that is 2-fold higher than the EBV RNA vaccine. In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a conventional vaccine at a dosage level that is 3-fold higher than the EBV RNA vaccine. In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a conventional vaccine at a dosage level that is 4-fold, 5-fold, 10-fold, 50-fold or 100-fold higher than the EBV RNA vaccine. In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a conventional vaccine at a dosage level that is 10-fold to 1000-fold higher than the EBV RNA vaccine. In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a conventional vaccine at a dosage level that is 100-fold to 1000-fold higher than the EBV RNA vaccine.

[0233] In other embodiments, the immune response is assayed by determination of the [protein] antibody titer in the subject. In other embodiments, the ability of serum or antibodies from an immunized subject is tested for the ability to neutralize virus uptake or reduce EBV transformation of human B lymphocytes. In other embodiments, the ability to enhance a robust T cell response(s) is measured using techniques recognized in the art.

[0234] In another aspect, the present disclosure provides a method of eliciting an immune response against Epstein - Barr virus (EBV) in a subject by administering to the subject an EBV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one EBV antigen, thereby inducing an immune response specific for the EBV antigen in the subject, wherein the immune response in the subject is induced 2 days to 10 weeks earlier compared to the immune response induced in a subject vaccinated with a conventional vaccine against EBV at a prophylactically effective dose. In some embodiments, the immune response in the subject is induced by a prophylactically effective dose of a conventional vaccine at a dosage level 2 - fold to 100 - fold higher than that of the RNA vaccine.

[0235] In some embodiments, the immune response in the subject is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks or 10 weeks earlier compared to the immune response induced in a subject vaccinated with a conventional vaccine at a prophylactically effective dose.

[0236] Also provided herein is a method of eliciting an immune response against EBV in a subject by administering to the subject an EBV RNA vaccine having an open reading frame encoding a first antigen, wherein the RNA polynucleotide does not contain a stabilizing element and the adjuvant is not co - formulated or co - administered with the vaccine.

[0237] An EBV RNA (e.g., mRNA) vaccine can be administered by any route that results in a therapeutically effective outcome. These include, but are not limited to, intradermal, intramuscular, intranasal, and / or subcutaneous administration. The present disclosure provides methods that include administering an RNA vaccine to a subject in need thereof. The exact amount required will vary between subjects depending on the species, age and general condition of the subject, the severity of the disease, the particular composition, the mode of administration, the mode of activity, as well as the same kind. EBV RNA (e.g., mRNA) vaccine compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of an EBV RNA (e.g., mRNA) vaccine composition can be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level, prophylactically effective dosage level, or appropriate imaging dosage level for any particular patient will depend on a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the age, weight, general health, sex and diet of the patient; the administration time, administration route and excretion rate of the specific compound used; the duration of the treatment; drugs used in combination with or simultaneously with the specific compound used; and like factors well known in the medical arts.

[0238] The effective amount of the EBV vaccine provided herein can be administered, for example, as a single dose of about 20 μg or as two 10 μg doses. In some embodiments, the effective amount is a total dose of 20 μg to 200 μg. For example, the effective amount can be a total dose of 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg or 200 μg. In some embodiments, the effective amount is a total dose of 25 μg to 200 μg. In some embodiments, the effective amount is a total dose of 50 μg to 200 μg.

[0239] In some embodiments, the EBV RNA (e.g., mRNA) vaccine composition is administered at a level sufficient to deliver a dosage of 0.0001 mg / kg to 100 mg / kg, 0.001 mg / kg to 0.05 mg / kg, 0.005 mg / kg to 0.05 mg / kg, 0.001 mg / kg to 0.005 mg / kg, 0.05 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 50 mg / kg, 0.1 mg / kg to 40 mg / kg, 0.5 mg / kg to 30 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.1 mg / kg to 10 mg / kg, or 1 mg / kg to 25 mg / kg of the subject's body weight per day, once or multiple times per day, per week, per month, etc., to obtain the desired therapeutic efficacy, diagnostic efficacy, prophylactic efficacy, or imaging efficacy (see, e.g., the unit dosage ranges described in International Publication WO2013078199, which is incorporated herein by reference in its entirety). The desired dosage will be delivered once every three days, once every two days, once a day, every other day, every three days, weekly, every two weeks, every three weeks, every four weeks, every two months, every three months, every six months, etc. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). When multiple administrations are used, a divided dosing regimen (such as those described herein) can be used. In an exemplary embodiment, the EBV RNA (e.g., mRNA) vaccine composition can be administered at a level sufficient to deliver a dosage of 0.0005 mg / kg to 0.01 mg / kg (e.g., from about 0.0005 mg / kg to about 0.0075 mg / kg, e.g., about 0.0005 mg / kg, about 0.001 mg / kg, about 0.002 mg / kg, about 0.003 mg / kg, about 0.004 mg / kg, or about 0.005 mg / kg).

[0240] In some embodiments, the EBV RNA (e.g., mRNA) vaccine composition can be administered one or two (or more) times at a level sufficient to deliver a dosage of 0.025 mg / kg to 0.250 mg / kg, 0.025 mg / kg to 0.500 mg / kg, 0.025 mg / kg to 0.750 mg / kg, or 0.025 mg / kg to 1.0 mg / kg.

[0241] In some embodiments, the EBV RNA (e.g., mRNA) vaccine composition can be administered two times (e.g., on day 0 and day 7, day 0 and day 14, day 0 and day 21, day 0 and day 28, day 0 and day 60, day 0 and day 90, day 0 and day 120, day 0 and day 150, day 0 and day 180, day 0 and 3 months later, day 0 and 6 months later, day 0 and 9 months later, day 0 and 12 months later, day 0 and 18 months later, day 0 and 2 years later, day 0 and 5 years later, or day 0 and 10 years later) at a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg, or at a dosage level sufficient to deliver those total doses. Higher dosages and frequencies of administration as well as lower dosages and frequencies of administration are encompassed by the present disclosure. For example, the EBV RNA (e.g., mRNA) vaccine composition can be administered three or four times.

[0242] In some embodiments, the EBV RNA (e.g., mRNA) vaccine composition can be administered in a total dose of 0.010 mg, 0.025 mg, 0.100 mg or 0.400 mg, or at a dosage level sufficient to deliver those total doses, two times (e.g., on day 0 and day 7, day 0 and day 14, day 0 and day 21, day 0 and day 28, day 0 and day 60, day 0 and day 90, day 0 and day 120, day 0 and day 150, day 0 and day 180, day 0 and 3 months later, day 0 and 6 months later, day 0 and 9 months later, day 0 and 12 months later, day 0 and 18 months later, day 0 and 2 years later, day 0 and 5 years later, or day 0 and 10 years later).

[0243] In some embodiments, an EBV RNA (e.g., mRNA) vaccine for use in a method of vaccinating a subject is administered to the subject in an amount effective to vaccinate the subject, which is a single dosage of the nucleic acid vaccine between 10 μg / kg and 400 μg / kg. In some embodiments, an RNA vaccine for use in a method of vaccinating a subject is administered to the subject in an amount effective to vaccinate the subject, which is a single dosage of the nucleic acid vaccine between 10 μg and 400 μg. In some embodiments, an EBV RNA (e.g., mRNA) vaccine for use in a method of vaccinating a subject is administered to the subject as a single dosage of 25 - 1000 μg (e.g., a single dosage of mRNA encoding an EBV antigen). In some embodiments, the EBV RNA vaccine is administered to the subject as a single dosage of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μg. For example, the EBV RNA vaccine can be administered to the subject as a single dose of 25 - 100, 25 - 500, 50 - 100, 50 - 500, 50 - 1000, 100 - 500, 100 - 1000, 250 - 500, 250 - 1000 or 500 - 1000 μg. In some embodiments, an EBV RNA (e.g., mRNA) vaccine for use in a method of vaccinating a subject is administered to the subject as two dosages, and the combination is equal to 25 - 1000 μg of the EBV RNA (e.g., mRNA) vaccine.

[0244] The EBV RNA (e.g., mRNA) vaccine pharmaceutical composition described herein can be formulated into the dosage forms described herein (intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal and subcutaneous), etc.).

[0245] Vaccine efficacy Some aspects of the present disclosure provide for the formulation of an EBV RNA (e.g., mRNA) vaccine, where the EBV RNA vaccine is formulated in an amount effective to produce an antigen-specific immune response in a subject (e.g., production of specific antibodies against EBV antigens). An “effective amount” is an amount of the EBV RNA (e.g., mRNA) vaccine effective to produce an antigen-specific immune response. Also provided herein is a method of inducing an antigen-specific immune response in a subject.

[0246] As used herein, an immune response to the vaccine or LNP of the invention is the development in a subject of a humoral immune response and / or a cellular immune response to the EBV protein(s) present in the vaccine. For purposes of the present invention, a “humoral” immune response refers to an immune response mediated by antibody molecules (including, e.g., secretory (IgA) molecules or IgG molecules), while a “cellular” immune response is an immune response mediated by T lymphocytes (e.g., CD4+ helper T cells and / or CD8+ T cells (e.g., CTLs)) and / or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytotoxic T cells (CTLs). CTLs have specificity for peptide antigens that are associated with and presented by proteins encoded by the major histocompatibility complex (MHC) and expressed on the surface of cells. CTLs assist in inducing and promoting the destruction of intracellular microorganisms or the lysis of cells infected with such microorganisms. Another aspect of cellular immunity involves an antigen-specific response by helper T cells. Helper T cells act to assist in stimulating and focusing the functions of non-specific effector cells against cells presenting peptide antigens in association with MHC molecules on their surface. A cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T cells and / or other white blood cells (including those derived from CD4+ T cells and CD8+ T cells).

[0247] In some embodiments, the antigen-specific immune response is characterized by measuring the anti-EBV antigen antibody titer produced in a subject inoculated with the EBV RNA (e.g., mRNA) vaccine provided herein. Antibody titer is a measure of the amount of antibody (e.g., an antibody specific for a particular antigen (e.g., anti-EBV antigen) or an epitope of an antigen) within a subject. Antibody titer is typically expressed as the reciprocal of the greatest dilution that gives a positive result. For example, enzyme-linked immunosorbent assay (ELISA) is a common assay for determination of antibody titer.

[0248] In some embodiments, antibody titer is used to assay whether a subject has an infection or to determine whether immunity is required. In some embodiments, antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective, and to identify any recent or previous infections. According to the present disclosure, antibody titer can be used to determine the strength of the immune response induced in a subject by an EBV RNA (e.g., mRNA) vaccine.

[0249] In some embodiments, the anti-EBV antigen antibody titer produced in a subject increases by at least 1 log-fold compared to a control. For example, the anti-EBV antigen antibody titer produced in a subject can increase by at least 1.5, at least 2, at least 2.5, or at least 3 log-fold compared to a control. In some embodiments, the anti-EBV antigen antibody titer produced in a subject increases by 1, 1.5, 2, 2.5, or 3 log-fold compared to a control. In some embodiments, the anti-EBV antigen antibody titer produced in a subject increases by 1-3 log-fold compared to a control. For example, the anti-EBV antigen antibody titer produced in a subject can increase by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log-fold compared to a control.

[0250] In some embodiments, the anti-EBV antigen antibody titer produced in the subject increases by at least 2-fold compared to the control. For example, the anti-EBV antigen antibody titer produced in the subject can increase by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold compared to the control. In some embodiments, the anti-EBV antigen antibody titer produced in the subject increases by 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold compared to the control. In some embodiments, the anti-EBV antigen antibody titer produced in the subject increases by 2 to 10-fold compared to the control. For example, the anti-EBV antigen antibody titer produced in the subject can increase by 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9 or 9 to 10-fold compared to the control.

[0251] In some embodiments, the control is the anti-EBV antigen antibody titer produced in a subject not inoculated with an EBV RNA (e.g., mRNA) vaccine. In some embodiments, the control is the anti-EBV antigen antibody titer produced in a subject administered a recombinant or purified EBV protein vaccine. Recombinant protein vaccines typically contain protein antigens produced in a heterologous expression system (e.g., bacteria or yeast) or purified from large amounts of pathogens.

[0252] In some embodiments, the ability of an EBV vaccine to be effective is measured in a mouse model. For example, an EBV vaccine is administered to a mouse model, and the mouse model can be assayed for induction of neutralizing antibody titers. Viral challenge studies can also be used to assess the effectiveness of the vaccines of the present disclosure. For example, an EBV vaccine is administered to a mouse model, the mouse model is challenged with EBV, and the mouse model can be assayed for survival and / or immune responses (e.g., neutralizing antibody response, T cell response (e.g., cytokine response)).

[0253] In some embodiments, the effective amount of an EBV RNA (e.g., mRNA) vaccine is a reduced dose compared to the standard therapeutic dose of a recombinant EBV protein vaccine. As used herein, "standard of care" refers to the guidelines for medical or psychological treatment and can be general or specific. The "standard of care" defines appropriate treatment based on the scientific evidence involved in the treatment of a given condition and the consensus among medical experts. This is the diagnostic and treatment process that a physician / clinician should follow for a particular type of patient, disease, or clinical situation. As used herein, "standard therapeutic dose" refers to the dose of a recombinant or purified EBV protein vaccine, or a live attenuated or inactivated EBV vaccine, or an EBV VLP vaccine, which a physician / clinician or other medical expert would administer to a subject while following the standard of care guidelines for the treatment or prevention of EBV (or EBV-related conditions).

[0254] In some embodiments, the anti-EBV antigen antibody titers produced in a subject administered an effective amount of an EBV RNA vaccine are equivalent to the anti-EBV antigen antibody titers produced in control subjects administered a standard therapeutic dose of a recombinant or purified EBV protein vaccine, a live attenuated or inactivated EBV vaccine, or an EBV VLP vaccine.

[0255] In some embodiments, the effective amount of the EBV RNA (e.g., mRNA) vaccine is an amount equivalent to a reduction to at least one-half of the standard therapeutic dose of a recombinant or purified EBV protein vaccine. For example, the effective amount of the EBV RNA vaccine can be an amount equivalent to a reduction to at least one-third, at least one-fourth, at least one-fifth, at least one-sixth, at least one-seventh, at least one-eighth, at least one-ninth, or at least one-tenth of the standard therapeutic dose of a recombinant or purified EBV protein vaccine. In some embodiments, the effective amount of the EBV RNA vaccine is an amount equivalent to a reduction to at least one-hundredth, at least one-five-hundredth, or at least one-thousandth of the standard therapeutic dose of a recombinant or purified EBV protein vaccine. In some embodiments, the effective amount of the EBV RNA vaccine is an amount equivalent to a reduction to one-half, one-third, one-fourth, one-fifth, one-sixth, one-seventh, one-eighth, one-ninth, one-tenth, one-twentieth, one-fiftieth, one-hundredth, one-two-hundred-and-fiftieth, one-five-hundredth, or one-thousandth of the standard therapeutic dose of a recombinant or purified EBV protein vaccine. In some embodiments, the anti-EBV antigen antibody titer produced in a subject to whom the effective amount of the EBV RNA vaccine has been administered is equivalent to the anti-EBV antigen antibody titer produced in a control subject to whom a recombinant or protein EBV protein vaccine at the standard therapeutic dose, a live attenuated or inactivated EBV vaccine, or an EBV VLP vaccine has been administered. In some embodiments, the effective amount of the EBV RNA (e.g., mRNA) vaccine is an amount equivalent to a reduction to one-half to one-thousandth (e.g., one-half to one-hundredth, one-tenth to one-thousandth) of the standard therapeutic dose of a recombinant or purified EBV protein vaccine, wherein the anti-EBV antigen antibody titer produced in the subject is equivalent to the anti-EBV antigen antibody titer produced in a control subject to whom a recombinant or purified EBV protein vaccine at the standard therapeutic dose, a live attenuated or inactivated EBV vaccine, or an EBV VLP vaccine has been administered.

[0256] In some embodiments, the effective amount of the EBV RNA (e.g., mRNA) vaccine is 2 to 1000, 2 to 900, 2 to 800, 2 to 700, 2 to 600, 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 1000, 3 to 900, 3 to 800, 3 to 700, 3 to 600, 3 to 500, 3 to 400, 3 to 300, 3 to 200, 3 to 100, 3 to 90, 3 to 80, 3 to 70, 3 to 60, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 1000, 4 to 900, 4 to 800, 4 to 700, 4 to 600, 4 to 500, 4 to 400, 4 to 300, 4 to 200, 4 to 100, 4 to 90, 4 to 80, 4 to 70, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 4 to 4, 5 to 1000, 5 to 900, 5 to 800, 5 to 700, 5 to 600, 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 1000, 6 to 900, 6 to 800, 6 to 700, 6 to 600, 6 to 500, 6 to 400, 6 to 300, 6 to 200, 6 to 100, 6 to 90, 6 to 80, 6 to 70, 6 to 60, 6 to 50, 6 to 40, 6 to 30, 6 to 20, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 1000, 7 to 900, 7 to 800, 7 to 700, 7 to 600, 7 to 500, 7 to 400, 7 to 300, 7 to 200, 7 to 100, 7 to 90, 7 to 80, 7 to 70, 7 to 60, 7 to 50, 7 to 40, 7 to 30, 7 to 20, 7 to 10, 7 to 9, 7 to 8, 8 to 1000, 8 to 900, 8 to 800, 8 to 700, 8 to 600, 8 to 500, 8 to 400, 8 to 300, 8 to 200, 8 to 100, 8 to 90, 8 to 80, 8 to 70, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 20, 8 to 10, 8 to 9, 9 to 1000, 9 to 900, 9 to 800, 9 to 700, 9 to 600, 9 to 500, 9 to 400, 9 to 300, 9 to 200, 9 to 100, 9 to 90, 9 to 80, 9 to 70,9~60、9~50、9~40、9~30、9~20、9~10、10~1000、10~900、10~800、10~700、10~600、10~500、10~400、10~300、10~200、10~100、10~90、10~80、10~70、10~60、10~50、10~40、10~30、10~20、20~1000、20~900、20~800、20~700、20~600、20~500、20~400、20~300、20~200、20~100、20~90、20~80、20~70、20~60、20~50、20~40、20~30、30~1000、30~900、30~800、30~700、30~600、30~500、30~400、30~300、30~200、30~100、30~90、30~80、30~70、30~60、30~50、30~40、40~1000、40~900、40~800、40~700、40~600、40~500、40~400、40~300、40~200、40~100、40~90、40~80、40~70、40~60、40~50、50~1000、50~900、50~800、50~700、50~600、50~500、50~400、50~300、50~200、50~100、50~90、50~80、50~70、50~60、60~1000、60~900、60~800、60~700、60~600、60~500、60~400、60~300、60~200、60~100、60~90、60~80、60~70、70~1000、70~900、70~800、70~700、70~600、70~500、70~400、70~300、70~200、70~100、70~90、70~80、80~1000、80~900、80~800、80~700、80~600、80~500、80~400、80~300、80~200、80~100、80~90、90~1000、90~900、90~800、90~700、90~600、90~500、90~400、90~300、90~200、90~100、100~1000、100~900、100~800、100~700、100~600、100~500、100~400、100~300、100~200、200~1000、200~900、200~800、200~700、200~600、Reductions to 1 / 200 to 1 / 500, 1 / 200 to 1 / 400, 1 / 200 to 1 / 300, 1 / 300 to 1 / 1000, 1 / 300 to 1 / 900, 1 / 300 to 1 / 800, 1 / 300 to 1 / 700, 1 / 300 to 1 / 600, 1 / 300 to 1 / 500, 1 / 300 to 1 / 400, 1 / 400 to 1 / 1000, 1 / 400 to 1 / 900, 1 / 400 to 1 / 800, 1 / 400 to 1 / 700, 1 / 400 to 1 / 600, 1 / 400 to 1 / 500, 1 / 500 to 1 / 1000, 1 / 500 to 1 / 900, 1 / 500 to 1 / 800, 1 / 500 to 1 / 700, 1 / 500 to 1 / 600, 1 / 600 to 1 / 1000, 1 / 600 to 1 / 900, 1 / 600 to 1 / 800, 1 / 600 to 1 / 700, 1 / 700 to 1 / 1000, 1 / 700 to 1 / 900, 1 / 700 to 1 / 800, 1 / 800 to 1 / 1000, 1 / 800 to 1 / 900, or 1 / 900 to 1 / 1000 are equivalent doses. In some embodiments (such as those described above), the anti-EBV antigen antibody titer produced in the subject is equivalent to the anti-EBV antigen antibody titer produced in control subjects administered a standard therapeutic dose of a recombinant or purified EBV protein vaccine, a live attenuated or inactivated EBV vaccine, or an EBV VLP vaccine. In some embodiments, the effective amount is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 1280, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000-fold reduction (or at least equivalent thereto) of the standard therapeutic dose of a recombinant EBV protein vaccine. In some embodiments (such as those described above), the anti-EBV antigen antibody titer produced in the subject is equivalent to the anti-EBV antigen antibody titer produced in control subjects administered a standard therapeutic dose of a recombinant or purified EBV protein vaccine,It is equivalent to the anti-EBV antigen antibody titer produced in control subjects administered with a live attenuated or inactivated EBV vaccine or an EBV VLP vaccine.

[0257] In some embodiments, the effective amount of the EBV RNA (e.g., mRNA) vaccine is a total dose of 50 to 1000 μg. In some embodiments, the effective amount of the EBV RNA (e.g., mRNA) vaccine is a total dose of 50 to 1000, 50 to 900, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 50 to 90, 50 to 80, 50 to 70, 50 to 60, 60 to 1000, 60 to 900, 60 to 800, 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 300, 60 to 200, 60 to 100, 60 to 90, 60 to 80, 60 to 70, 70 to 1000, 70 to 900, 70 to 800, 70 to 700, 70 to 600, 70 to 500, 70 to 400, 70 to 300, 70 to 200, 70 to 100, 70 to 90, 70 to 80, 80 to 1000, 80 to 900, 80 to 800, 80 to 700, 80 to 600, 80 to 500, 80 to 400, 80 to 300, 80 to 200, 80 to 100, 80 to 90, 90 to 1000, 90 to 900, 90 to 800, 90 to 700, 90 to 600, 90 to 500, 90 to 400, 90 to 300, 90 to 200, 90 to 100, 100 to 1000, 100 to 900, 100 to 800, 100 to 700, 100 to 600, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 200 to 1000, 200 to 900, 200 to 800, 200 to 700, 200 to 600, 200 to 500, 200 to 400, 200 to 300, 300 to 1000, 300 to 900, 300 to 800, 300 to 700, 300 to 600, 300 to 500, 300 to 400, 400 to 1000, 400 to 900, 400 to 800, 400 to 700, 400 to 600, 400 to 500, 500 to 1000, 500 to 900, 500 to 800, 500 to 700, 500 to 600, 600 to 1000, 600 to 900, 600 to 900, 600 to 700, 700 to 1000, 700 to 900, 700 to 800, 800 to 1000, 800 to 900, or 900 to 1000 μg.In some embodiments, the effective amount of the EBV RNA (e.g., mRNA) vaccine is a total dose of 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μg. In some embodiments, the effective amount is a dose of 25 - 500 μg administered to the subject a total of 2 times. In some embodiments, the effective amount of the EBV RNA (e.g., mRNA) vaccine is a dose of 25 - 500, 25 - 400, 25 - 300, 25 - 200, 25 - 100, 25 - 50, 50 - 500, 50 - 400, 50 - 300, 50 - 200, 50 - 100, 100 - 500, 100 - 400, 100 - 300, 100 - 200, 150 - 500, 150 - 400, 150 - 300, 150 - 200, 200 - 500, 200 - 400, 200 - 300, 250 - 500, 250 - 400, 250 - 300, 300 - 500, 300 - 400, 350 - 500, 350 - 400, 400 - 500 or 450 - 500 μg administered to the subject a total of 2 times. In some embodiments, the effective amount of the EBV RNA (e.g., mRNA) vaccine is a total dose of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 μg administered to the subject a total of 2 times.

[0258] Vaccine efficacy can be assayed using standard assays (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1;201(11):1607-10). For example, vaccine efficacy can be measured by a double-blind randomized controlled clinical trial. Vaccine efficacy is expressed as a proportional reduction in the incidence of disease (AR) between a study cohort without vaccination (ARU) and a study cohort with vaccination (ARV), and can be calculated from the relative risk (RR) of disease in the vaccinated group using the following formula. Efficacy = (ARU - ARV) / ARU × 100; and Efficacy = (1 - RR) × 100.

[0259] Similarly, vaccine effectiveness can be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1;201(11):1607-10). Vaccine effectiveness is an assessment of how a vaccine (which may already have been shown to have high vaccine efficacy) reduces disease in a population. This measurement can assess the net balance of benefits and side effects of the vaccine itself as well as the vaccination program under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy, but is also affected by how well the target group in the population is immunized, as well as by other vaccine-unrelated factors that affect "real-world" outcomes such as hospitalizations, clinic visits, or costs. For example, a retrospective case-control analysis is used, where the rates of vaccination among infectious cases and a suitable set of controls are compared. Vaccine effectiveness can be expressed as a rate difference using the odds ratio (OR) for developing an infection despite vaccination. Effectiveness = (1 - OR) × 100.

[0260] In some embodiments, the effectiveness of the EBV vaccine is at least 60% compared to unvaccinated control subjects. For example, the effectiveness of the EBV vaccine can be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100% compared to unvaccinated control subjects.

[0261] Sterilizing immunity. Sterilizing immunity refers to a unique immune state that prevents effective pathogen infection into the host. In some embodiments, an effective amount of the EBV vaccine of the present disclosure is sufficient to provide sterilizing immunity in a subject for at least one year. For example, an effective amount of the EBV vaccine of the present disclosure is sufficient to provide sterilizing immunity in a subject for at least two years, at least three years, at least four years or at least five years. In some embodiments, an effective amount of the EBV vaccine of the present disclosure is sufficient to provide sterilizing immunity in a subject at a dose that is at least one-fifth lower compared to a control. For example, the effective amount may be sufficient to provide sterilizing immunity in a subject at a dose that is at least one-tenth, one-fifteenth or one-twentieth lower compared to a control.

[0262] Detectable antigen. In some embodiments, an effective amount of the EBV vaccine of the present disclosure is sufficient to produce a detectable level of EBV antigen when measured in the serum of a subject 1 to 72 hours after administration.

[0263] Titer. Antibody titer is a measurement of the amount of antibody (e.g., an antibody specific to a particular antigen (e.g., an anti-EBV antigen)) within a subject. Antibody titer is typically expressed as the reciprocal of the greatest dilution that gives a positive result. For example, enzyme-linked immunosorbent assay (ELISA) is a common assay for the determination of antibody titer.

[0264] In some embodiments, an effective amount of the EBV vaccine of the present disclosure is sufficient to produce a neutralizing antibody titer of 1,000 to 10,000 produced by neutralizing antibodies against EBV antigen when measured in the serum of a subject 1 to 72 hours after administration. In some embodiments, the effective amount is sufficient to produce a neutralizing antibody titer of 1,000 to 5,000 produced by neutralizing antibodies against EBV antigen when measured in the serum of a subject 1 to 72 hours after administration. In some embodiments, the effective amount is sufficient to produce a neutralizing antibody titer of 5,000 to 10,000 produced by neutralizing antibodies against EBV antigen when measured in the serum of a subject 1 to 72 hours after administration.

[0265] In some embodiments, the neutralizing antibody titer is at least 100 NT 50 For example, the neutralizing antibody titer can be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NT 50 In some embodiments, the neutralizing antibody titer is at least 10,000 NT 50 is.

[0266] In some embodiments, the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU / mL). For example, the neutralizing antibody titer can be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NU / mL. In some embodiments, the neutralizing antibody titer is at least 10,000 NU / mL.

[0267] In some embodiments, the anti-EBV antigen antibody titer produced in a subject increases by at least 1 log fold compared to a control. For example, the anti-EBV antigen antibody titer produced in a subject can increase by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 log fold compared to a control.

[0268] In some embodiments, the anti-EBV antigen antibody titer produced in a subject increases by at least 2-fold compared to a control. For example, the anti-EBV antigen antibody titer produced in a subject increases by at least 3, 4, 5, 6, 7, 8, 9 or 10-fold compared to a control.

[0269] In some embodiments, the geometric mean (which is the nth root of the product of n numbers) is generally used to describe a proportional increase. The geometric mean is used in some embodiments to characterize the antibody titer produced in a subject.

[0270] The control can be, for example, a subject who has not been vaccinated, or a subject who has been administered a live attenuated EBV vaccine, an inactivated EBV vaccine, or a protein subunit EBV vaccine.

Example

[0271] Example 1 EBV glycoprotein 350 (gp350) variants were produced and their expression was tested in HeLa cells. HeLa cells were transiently transfected for 24 hours with mRNA (SEQ ID NOs: 185, 182, 207, and 208) encoding each of the EBV glycoprotein 350 (gp350) variants. Flow cytometry analysis (Figure 1A, 1 μg dose of mRNA) and immunoassay (Figure 1B, 1 μg dose of mRNA; Figure 1C, 0.5 μg dose of mRNA) using an EBV neutralizing antibody ("72A1") that binds to the conformational epitope in gp350 demonstrate that all EBV gp350 variants tested show equivalent expression on the surface of transfected HeLa cells. Unless otherwise specified, all mRNA vaccines can be formulated in lipid nanoparticles containing Compound 1 lipids, such as 20 - 60% ionizable cationic lipids, 5 - 25% non-cationic lipids, 25 - 55% sterols, and 0.5 - 15% PEGylated lipids.

[0272] Example 2 Sequences of EBV gp350 mRNA (SEQ ID NOs: 81 and 204) with two different 5' UTR sequences were produced and their expression was tested in HeLa cells. HeLa cells were transiently transfected for 24 hours with 0.5 μg dose of mRNA encoding the EBV gp350 antigen. Flow cytometry analysis (Figure 2A) and immunoassay (Figure 2B) using an anti-72A1 antibody demonstrate that both EBV gp350 mRNA constructs tested show equivalent expression on the surface of transfected HeLa cells.

[0273] Example 3 Balb / c mice were vaccinated intramuscularly with an EBV vaccine containing mRNA encoding EBV gp350 variants (SEQ ID NOs: 185, 182, 207, and 208) formulated in lipid nanoparticles. A dose of 2 μg was administered on day 1 and again on day 22. Blood was collected from the mice on days 21 and 43. The results demonstrate that all EBV gp350 vaccines tested induced gp350-specific IgG antibody titers in serum at day 21 (3 weeks after prime) and day 43 (3 weeks after boost) (Figure 3).

[0274] Example 4 Additional EBV antigens and antigen complexes were produced and their expression was tested in HeLa cells. HeLa cells were transiently transfected for 24 hours with 0.25 μg of (1) mRNA encoding EBV gH (SEQ ID NO: 187), mRNA encoding EBV gL (SEQ ID NO: 188), and mRNA encoding EBV gp42 (SEQ ID NO: 189) (EBV gH / gL / gp42 UTR A); (2) mRNA encoding EBV gH (SEQ ID NO: 201), mRNA encoding EBV gL (SEQ ID NO: 202), and mRNA encoding EBV gp42 (SEQ ID NO: 203) (EBV gH / gL / gp42 UTR B); (3) mRNA encoding EBV gH (SEQ ID NO: 187) and mRNA encoding gp42 (SEQ ID NO: 189); or (4) mRNA encoding EBV gp42 (SEQ ID NO: 189). Flow cytometry analysis (Figure 4A) and immunoassay (Figure 4B) using anti-gH / gL / gp42 (2D4) antibody demonstrate that all EBV mRNA constructs tested show equivalent expression on the surface of transfected HeLa cells.

[0275] Example 5 Balb / c mice were vaccinated intramuscularly with an EBV vaccine containing (1) mRNA encoding EBV gH (SEQ ID NO: 187), mRNA encoding EBV gL (SEQ ID NO: 188), and mRNA encoding EBV gp42 (SEQ ID NO: 189); (2) mRNA encoding EBV gp350 (SEQ ID NO: 185); or (3) mRNA encoding EBV gH (SEQ ID NO: 187), mRNA encoding EBV gL (SEQ ID NO: 188), mRNA encoding EBV gp42 (SEQ ID NO: 189), and mRNA encoding EBV gp350 (SEQ ID NO: 185). A dose of 2 μg was administered on day 1 and again on day 29. Blood was collected from the mice on days 28 and 57. The results demonstrate that all EBV vaccines tested induced neutralizing antibody titers at day 28 (4 weeks after priming) and day 57 (4 weeks after boosting) post-vaccination (Figure 5).

[0276] Example 6 HeLa cells were transiently transfected for 24 hours with 0.5 μg of (1) mRNA encoding EBV gH (SEQ ID NO: 201) and mRNA encoding EBV gL (SEQ ID NO: 202); or (2) mRNA encoding only EBV gH (SEQ ID NO: 201). Flow cytometry analysis using anti-gH / gL (2A8) antibody or anti-gH / gL / gp42 (2D4) antibody demonstrates that the 2A8 antibody specifically binds to EBV gL expressed on the surface of HeLa cells (compared to the data presented in Example 4). Immunassays after 24-hour transfection with either 0.25 μg of (1) mRNA encoding EBV mRNA encoding EBV gH (SEQ ID NO: 187) and mRNA encoding EBV gL (SEQ ID NO: 188); or (2) mRNA encoding EBV mRNA encoding EBV gH (SEQ ID NO: 201) and mRNA encoding EBV gL (SEQ ID NO: 202) show comparable expression of both EBV antigen complexes on the surface of HeLa cells.

[0277] Example 7 Three additional immunogenicity studies were conducted using various mRNA vaccines of the present disclosure (formulated in lipid nanoparticles) and various antigen-specific antibodies. Balb / c mice were vaccinated intramuscularly with (1) mRNA encoding EBV gH (SEQ ID NO: 187) and mRNA encoding EBV gL (SEQ ID NO: 188) at a dose of 5 μg or 1 μg; (2) mRNA encoding EBV gH (SEQ ID NO: 187), mRNA encoding EBV gL (SEQ ID NO: 188), and mRNA encoding EBV gB (SEQ ID NO: 209) at a dose of 7.5 μg or 1.5 μg; (3) mRNA encoding EBV gH (SEQ ID NO: 187), mRNA encoding EBV gL (SEQ ID NO: 188), and mRNA encoding EBV gp350 (SEQ ID NO: 185) at a dose of 7.5 μg or 1.5 μg; or (4) mRNA encoding EBV gH (SEQ ID NO: 187), mRNA encoding EBV gL (SEQ ID NO: 188), mRNA encoding EBV gB (SEQ ID NO: 209), and mRNA encoding EBV gp350 (SEQ ID NO: 185) at a dose of 10 μg or 2 μg. Blood was collected from the mice on day 57. Results following detection with anti-gH / gL antibodies (Figure 7), anti-gB antibodies (Figure 8), or anti-gp350 antibodies (Figure 9) demonstrate the induction of EBV antigen-specific neutralizing antibodies.

[0278] Example 8 Mice were vaccinated intramuscularly with mRNA encoding one of four EBV latency genes (LMP1 (SEQ ID NO: 179), LMP2 (SEQ ID NO: 181), EBNA1 Δ1-400 (SEQ ID NO: 178) or EBNA3A (SEQ ID NO: 177)) or all combinations of the four mRNA vaccines (LMP1 (SEQ ID NO: 179), LMP2 (SEQ ID NO: 181), EBNA1 Δ1-400(Accession No.: 178) and EBNA3A (Accession No.: 177)) were vaccinated. Cells were harvested from vaccinated mice and stimulated with LMP1, LMP2, EBNA1 or EBNA3A peptides. All peptide libraries contain 15-mer peptides that overlap by 11 amino acids. CD8 T cell responses are shown in FIGS. 10A-10D, and CD4 T cell responses are shown in FIGS. 11A-11D.

[0279] Example 9 mRNA (gH-gL) ligation constructs (SEQ ID NO: 218 or 221) encoding EBV glycoprotein H-glycoprotein L were produced and their expression was tested in HeLa cells. HeLa cells were transiently transfected for 24 hours with mRNA (SEQ ID NO: 218 or SEQ ID NO: 221) encoding two EBV gH-gL variants with different linkers, or mRNA encoding EBV gH (EBV gH mRNA; SEQ ID NO: 228) and mRNA encoding EBV gL (EBV gL mRNA; SEQ ID NO: 229). Flow cytometry analysis (FIG. 13A, 0.5 μg dose of EBV gH-gL ligation mRNA, or 0.25 μg dose of EBV gH mRNA and EBV gL mRNA each) and immunoassay (FIG. 13B; 0.5 μg dose of gH-gL ligation construct, or 0.25 μg dose of gH and gL each) using EBV neutralizing antibodies 2A8 and CL40 that bind to conformational epitopes in gH demonstrate that both EBV gH-gL constructs (SEQ ID NO: 218 and 221) tested are expressed on the surface of transfected HeLa cells.

[0280] Example 10 In further immunogenicity studies to test the effects of different untranslated regions (UTRs), mice were vaccinated intramuscularly with: (1) 10 μg of mRNA encoding gp350 (SEQ ID NO: 185), mRNA encoding gH (SEQ ID NO: 187), mRNA encoding gL (SEQ ID NO: 188), mRNA encoding the LMP2 antigen (SEQ ID NO: 181) and mRNA encoding the EBNA1 antigen (SEQ ID NO: 178) (each of the transcripts contains UTRA); (2) 10 μg of mRNA encoding gp350 (SEQ ID NO: 185), mRNA encoding gH (SEQ ID NO: 187), mRNA encoding gL (SEQ ID NO: 188), mRNA encoding the LMP2 antigen (SEQ ID NO: 181) and mRNA encoding the EBNA1 antigen (SEQ ID NO: 178) (each of the transcripts contains UTRB); (3) 2 μg of mRNA encoding the EBNA1 antigen (SEQ ID NO: 178) with UTRB; (4) 2 μg of mRNA encoding the LMP2 antigen with UTRB; or (5) lipid nanoparticles (Compound 1 lipids, e.g., containing 20 - 60% ionizable cationic lipid, 5 - 25% non-cationic lipid, 25 - 55% sterol and 0.5 - 15% PEG-modified lipid) containing empty lipid nanoparticles. Mice were administered one dose on day 1 and a second dose on day 29. Blood samples were taken immediately prior to dosing and on day 57. Spleens were collected from a subset of the animals on day 36. Cells were harvested from vaccinated mice and stimulated with peptides from an EBNA1 peptide library (Figure 14) or an LMP2 library (Figure 15). The peptide libraries contained 15mer peptides that overlapped by 11 amino acids. Cytokine responses of CD4 T cells for the EBV formulation (Groups 1 and 2), EBNA1 formulation (Group 3) and control (empty nanoparticles; Group 5) are shown in the upper row of Figure 14. The lower row of Figure 14 shows the CD8 T cell response. Figure 15 shows the CD4 T cell response (upper row) and CD8 T cell cytokine response (lower row) for the EBV formulation (Groups 1 and 2), LMP2 formulation (Group 4) and control (empty nanoparticles; Group 5). Similar T cell responses were found among the groups regardless of the UTR used.

[0281] Example 11 The construct was tested in a non - human primate (Rhesus macaque) model. Subjects were vaccinated intramuscularly with lipid nanoparticles containing (1) 200 μg of mRNA encoding gp350 (SEQ ID NO: 185), mRNA encoding gH (SEQ ID NO: 187), mRNA encoding gL (SEQ ID NO: 188), mRNA encoding LMP2 antigen (SEQ ID NO: 181), and mRNA encoding EBNA1 antigen (SEQ ID NO: 178); (2) 50 μg of mRNA encoding gp350 (SEQ ID NO: 185), mRNA encoding gH (SEQ ID NO: 187), mRNA encoding gL (SEQ ID NO: 188), mRNA encoding LMP2 antigen (SEQ ID NO: 181) and mRNA encoding EBNA1 antigen (SEQ ID NO: 178); or (3) 200 μg of mRNA encoding a control. As shown in Figure 16, subjects were administered one dose on day 1 and a second dose on day 28. Blood samples were taken on days 0, 27, 28, 60, 90, 120, 150 and 180. The results after detection of anti - gp350 titers and anti - gH / gL antibodies demonstrate that vaccination with the selected formulation results in increased and persistent titers of anti - gp350 and anti - gH / gL antibodies (Figure 16). Neutralizing antibody titers were found to be persistent at high doses, but a significant decline in neutralizing antibody titers at low doses of the EBV vaccine was observed (Figure 17).

[0282] Example 12 The effects of various downstream processes on EBV vaccine immunogenicity were investigated. EBV vaccines containing gp350 (SEQ ID NO: 185), gH (SEQ ID NO: 187) and gL (SEQ ID NO: 188) were synthesized using different downstream purification processes.

[0283] Dose formulations were formed using the resulting mRNA (10 μg, 3 μg, and 1 μg doses for each of the four groups; PBS was used as the control group; n = 8 / group), and they were administered to mice on days 1 and 22. Blood samples were collected on days 1, 21, 22, 36, 82, and 142. Antibody titers against gp250 (Figure 18) and gH / gL (Figure 19) were measured.

[0284] equivalent All references, patents, and patent applications disclosed herein are incorporated by reference in their entirety for the subject matter for which each is cited, and in some cases, they may encompass the entire document.

[0285] It should be understood that the indefinite articles "a" and "an," as used herein in the specification and claims, mean "at least one" unless clearly indicated to the contrary. It should also be understood that in any method claimed herein that includes two or more steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited, unless clearly indicated to the contrary.

[0286] In the claims, as well as in the specification above, all transitional phrases such as "comprising", "including", "carrying", "having", "containing", "involving", "holding", "consisting of", and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are to be considered as closed or semi-closed transitional phrases, as set forth in the United States Patent Office Manual of Patent Examining Procedures (Section 2111.03).

[0287] The terms "about" and "substantially" preceding a numerical value mean ±10% of the recited numerical value.

[0288] Where a range of values is provided, each value between the upper and lower limits of that range is specifically contemplated and described herein.

[0289] The entire contents of the international applications PCT / US2015 / 02740, PCT / US2016 / 043348, PCT / US2016 / 043332, PCT / US2016 / 058327, PCT / US2016 / 058324, PCT / US2016 / 058314, PCT / US2016 / 058310, PCT / US2016 / 058321, PCT / US2016 / 058297, PCT / US2016 / 058319, and PCT / US2016 / 058314 are hereby incorporated by reference into this specification.

[0290] Array list It should be understood that any of the mRNA sequences described herein may include a 5’ UTR and / or a 3’ UTR. The UTR sequence may be selected from the following sequences, or other known UTR sequences may be used. It should also be understood that any of the mRNA constructs described herein may further include a polyA tail and / or a cap (e.g., 7mG(5’)ppp(5’)NlmpNp). Furthermore, many of the mRNAs and encoded antigen sequences described herein include a signal peptide and / or a peptide tag (e.g., a C-terminal His tag), but it should be understood that the indicated signal peptide and / or peptide tag may be replaced with a different signal peptide and / or peptide tag, or the signal peptide and / or peptide tag may be omitted.

[0291] 5’ UTR: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 1) 5’ UTR: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACC (SEQ ID NO: 104) 3’ UTR: UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 3) 3’ UTR: UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 106)

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Claims

1. 1. An Epstein-Barr Virus (EBV) vaccine comprising a ribonucleic acid (RNA) having an open reading frame (ORF) encoding an EBV antigen, wherein intramuscular (IM) administration of a therapeutically effective amount of the vaccine to a subject induces neutralizing antibody titers and / or a T cell immune response in the subject.

2. 2. The vaccine of claim 1, wherein the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU / mL).

3. 3. The vaccine of claim 2, wherein the neutralizing antibody titer is at least 500 NU / mL.

4. 4. The vaccine of claim 3, wherein the neutralizing antibody titer is at least 1000 NU / mL.

5. 5. The vaccine of any one of claims 1 to 4, wherein the neutralizing antibody titer is sufficient to reduce EBV infection of B cells by at least 50% compared to neutralizing antibody titers in unvaccinated control subjects or compared to neutralizing antibody titers in subjects vaccinated with a live attenuated EBV vaccine, an inactivated EBV vaccine or a protein subunit EBV vaccine.

6. The vaccine of any one of claims 1 to 5, wherein the neutralizing antibody titers are induced in the subject after less than 3 doses of the vaccine.

7. The vaccine of any one of claims 1 to 6, wherein the single dose is between 10 μg and 100 μg.

8. 8. The vaccine of any one of claims 1 to 7, wherein the neutralizing antibody titers and / or T cell immune response are sufficient to reduce the rate of symptomatic infectious mononucleosis compared to the neutralizing antibody titers in unvaccinated control subjects.

9. 9. The vaccine of any one of claims 1 to 8, wherein the neutralizing antibody titers and / or T cell immune response are sufficient to reduce the rate of asymptomatic EBV infection compared to the neutralizing antibody titers in unvaccinated control subjects.

10. The vaccine of any one of claims 1 to 9, wherein the neutralizing antibody titers and / or T cell immune response are sufficient to prevent EBV latency in the subject.

11. The vaccine of any one of claims 1 to 10, wherein the neutralizing antibody titers are sufficient to block fusion of EBV with epithelial cells and / or B cells of the subject.

12. The vaccine of any one of claims 1 to 11, wherein the neutralizing antibody titers are induced within 20 days after a single dose of 10 to 100 μg of the vaccine.

13. The vaccine of any one of claims 1 to 12, wherein the neutralizing antibody titers are induced within 40 days after a second 10-100 μg dose of the vaccine.

14. The T cell immune response is ＋ The vaccine of any one of claims 1 to 13, which induces a T cell immune response.

15. The T cell immune response is CD8 ＋ The vaccine of any one of claims 1 to 14, which induces a T cell immune response.

16. The vaccine of any one of claims 1 to 15, wherein the EBV antigen is expressed on the surface of cells of the subject.

17. A single dose of 2 μg of the vaccine provides approximately 100 NT ５０ The vaccine of any one of claims 1 to 16, which induces neutralizing antibody titers in mice.

18. A booster dose of 2 μg of the vaccine ５０ 18. The vaccine of claim 17, which induces neutralizing antibody titers in mice.

19. The EBV vaccine comprises: (a) a ribonucleic acid (RNA) having an open reading frame (ORF) encoding two EBV antigens, or (b) two RNAs, each carrying an ORF encoding an EBV antigen; The vaccine according to any one of claims 1 to 18, comprising:

20. The vaccine of any one of claims 1 to 19, wherein the vaccine comprises one RNA with ORFs encoding two EBV antigens formulated in lipid nanoparticles.

21. The vaccine of any one of claims 1 to 19, wherein the vaccine comprises two RNAs, each having an ORF encoding an EBV antigen, and the two RNAs are formulated in a single lipid nanoparticle.

22. The vaccine of any one of claims 1 to 19, wherein the vaccine comprises two RNAs, each having an ORF encoding an EBV antigen, each of the RNAs being formulated in a single lipid nanoparticle.

23. The vaccine of any one of claims 1 to 22, further comprising at least one additional RNA having an ORF encoding at least one additional EBV antigen.

24. 24. The vaccine of any one of claims 20 to 23, wherein the lipid nanoparticles comprise, in molar ratio, 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

25. The vaccine of any one of claims 1 to 24, wherein the EBV antigen is selected from the group consisting of gp350, gH, gL, gB, gp42, LMP1, LMP2, EBNA1 and EBNA3.

26. 26. The vaccine of claim 25, wherein the EBV antigens comprise an EBV gp350 antigen, an EBV gH antigen and an EBV gL antigen, optionally, the EBV gH antigen is linked to the EBV gL antigen, optionally, the linker comprises a GGGGS motif, and optionally, the linker comprises the amino acid sequence of SEQ ID NO: 224 or SEQ ID NO:

225.

27. 27. The vaccine of claim 26, wherein the EBV antigens further comprise an EBV gp42 antigen and / or a gB antigen.

28. The vaccine of any one of claims 25 to 27, wherein the EBV gp350 antigen is a wild-type EBV gp350 antigen, a mutated EBV gp350 antigen or a truncated EBV gp350 antigen.

29. 29. The vaccine of any one of claims 1 to 28, wherein the RNA comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 201, 202, 203, 204, 207, 208, 177, 178, 179, 181, 182, 185, 187, 188, 189, 209, 218 and 221.

30. The vaccine of any one of claims 1 to 29, wherein the EBV antigen is fused to a scaffold moiety.

31. 31. The vaccine of claim 30, wherein the scaffold moiety is selected from the group consisting of ferritin, encapsulin, lumazine synthase, hepatitis B surface antigen, and hepatitis B core antigen.

32. The vaccine of any one of claims 1 to 31, wherein the RNA comprises messenger RNA (mRNA).

33. The vaccine of any one of claims 1 to 32, wherein the RNA further comprises a 5'UTR.

34. 34. The vaccine of claim 33, wherein the 5'UTR comprises a sequence identified by SEQ ID NO:1 or SEQ ID NO:

104.

35. The vaccine of any one of claims 1 to 34, wherein the RNA further comprises a 3'UTR.

36. 36. The vaccine of claim 35, wherein the 3'UTR comprises a sequence identified by SEQ ID NO:3 or SEQ ID NO:

106.

37. The vaccine of any one of claims 1 to 36, wherein the EBV antigen is fused to a signal peptide.

38. 38. The vaccine of claim 37, wherein the signal peptide is a bovine prolactin signal peptide, optionally comprising SEQ ID NO:

115.

39. The vaccine of any one of claims 1 to 38, wherein the RNA is unmodified.

40. The vaccine of any one of claims 1 to 38, wherein the RNA comprises at least one modified nucleotide.

41. 41. The vaccine of claim 40, wherein at least 80% of the uracils in the ORF contain 1-methyl-pseudouridine modifications.

42. A method comprising administering to a subject a therapeutically effective amount of an EBV vaccine according to any one of claims 1 to 41 to induce neutralizing antibody titers and / or a T cell immune response in said subject.

43. 43. The method of claim 42, wherein the efficacy of the EBV vaccine is at least 80% compared to unvaccinated control subjects.

44. 44. The method of claim 42 or 43, wherein detectable levels of EBV antigen are produced in the serum of the subject 1 to 72 hours after administration of the vaccine.

45. 45. The method of any one of claims 42 to 44, wherein a neutralizing antibody titer of at least 100 NU / ml is produced in the serum of the subject 1 to 72 hours after administration of the vaccine.

46. 46. ​​The method of claim 45, wherein a neutralizing antibody titer of at least 500 NU / ml is produced in the serum of the subject 1 to 72 hours after administration of the vaccine.

47. 47. The method of claim 46, wherein a neutralizing antibody titer of at least 1000 NU / ml is produced in the serum of the subject 1 to 72 hours after administration of the vaccine.

48. 48. The method of any one of claims 42-47, wherein the therapeutically effective amount is a total dose of 20 μg to 200 μg.

49. 49. The method of claim 48, wherein the therapeutically effective amount is a total dose of 50 μg to 100 μg.

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