Eb v vaccines and antibodies

Abstract

The present invention relates in particular to a composition comprising an Epstein-Barr virus (EBV) BILF2 glycoprotein, a nucleic acid encoding an EBV BILF2 glycoprotein, or an extracellular vesicle (EV) or an Epstein-Barr virus-like particle (VLP) displaying on its surface an EBV BILF2 glycoprotein, for use in a method of eliciting antibodies against said BILF2 glycoprotein that reduce the infectivity of EBV on human epithelial cells. Preferably, said composition elicits antibodies that prevent the infection of EBV on human epithelial cells.

Description

[0001] I. Technical Field

[0002] This invention particularly relates to a composition comprising the BILF2 glycoprotein of Epstein-Barr virus (EBV), nucleic acid encoding the BILF2 glycoprotein of EBV, or extracellular vesicles (EVs) or EBV virus-like particles (VLPs) displaying the BILF2 glycoprotein of EBV on their surface, wherein the composition is used in a method of inducing an antibody against the BILF2 glycoprotein, the antibody reducing the infectivity of EBV to human epithelial cells. Preferably, the composition induces an antibody that prevents EBV infection of human epithelial cells.

[0003] II. Background Technology

[0004] Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that infects over 90% of the global population and persists in its host throughout life. In most cases, primary infection occurs in early childhood and is usually asymptomatic. In contrast, if infection is delayed and occurs in adolescence or adulthood, it is usually symptomatic, causing a benign lymphoproliferative syndrome called infectious mononucleosis (IM) in up to 50% of cases. Although the disease is usually self-limiting, there have been reports of persistent IM, a common and complex disease called myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS), or a rare chronic active EBV infection (CAEBV) with fatal outcomes. Clinically significant IM has also been found to significantly increase the risk of later-life Hodgkin's disease and other types of lymphoma. IM is now also widely considered an independent risk factor for multiple sclerosis. In addition, EBV has a causal relationship with a heterogeneous group of malignant diseases, such as nasopharyngeal carcinoma, gastric cancer and various types of lymphoma, so the WHO has classified EBV as a Group 1 carcinogen.

[0005] In addition to the aforementioned medical conditions caused by EBV, patients with primary or secondary immunodeficiency (such as transplant recipients) are at high risk of EBV-related diseases due to the detrimental effects of immunosuppressants on the immune control of EBV-infected B cells. EBV-associated post-transplant lymphoproliferative disorder (PTLD) is a significant form of post-transplant complication, occurring in up to 20% of organ recipients. Importantly, immunocompromised transplant recipients who are immunologically naive to EBV at the onset of immunosuppression are at particularly high risk of developing life-threatening EBV+PTLD due to primary EBV infection (e.g., often due to high prevalence of EBV, transmitted through donor organs after transplantation). These patients cannot effectively elicit EBV-specific T cells, which play a crucial role in controlling the proliferation of EBV-infected B cells, due to impaired T-cell immunity resulting from exposure to immunosuppressive drugs. In contrast, patients who are seropositive for EBV at transplantation have a much lower risk of developing PTLD, demonstrating the vital role of EBV-specific T cells, which are ready to eliminate virus-infected cells and control EBV. Typically, patients who are pre-transplant EBV seronegative have a much higher risk of developing EBV-related diseases because transmission of donor EBV into the transplanted organ or natural infection with the virus can cause lymphoproliferative disorders in EBV seronegative recipients post-transplant. As with many virus-related diseases, vaccination is a promising approach to prevent and / or treat viral infection and its consequences in the host, and this is also true for reducing the high risk of PTLD in seronegative patients by immunizing them against EBV pre-transplant.

[0006] The first human trials of candidate vaccines were conducted as early as the 1990s, using recombinant vaccinia virus expressing the major EBV membrane antigen gp350 / 220, which produced elevated EBV neutralizing antibody titers. The gp350 / 220 glycoprotein is encoded by the viral BLLF1 gene. In recent years, various peptide-based prophylactic vaccines have been described, designed to achieve seroconversion and IM prevention in healthy volunteers and children awaiting kidney transplantation. Neutralizing antibodies were detected in four recipients of these children. However, the immune response declined rapidly and had little effect on post-transplant events.

[0007] EBV is a ubiquitous human herpesvirus that infects over 90% of the global population and persists in its host throughout life. Although initial infection in early childhood often causes self-limiting, mild illness or is completely asymptomatic, EBV infection is linked to the pathogenesis of multiple forms of cancer and multiple sclerosis and can trigger more severe disease in patients with primary or secondary immunodeficiency. These EBV-related diseases provide a strong basis for developing and improving methods to reduce EBV infectivity, which should safely and effectively address subsequent viral infections and related diseases.

[0008] However, limited evidence regarding persistent EBV infection and immune selection based on viral antigen variants suggests that in vivo neutralization of EBV infection is ineffective. Therefore, the technical problem of this invention is to provide an EBV vaccine capable of controlling EBV infectivity and thus controlling EBV infection.

[0009] The technical problem is solved by the subject matter defined in the claims, described in the specification, and shown in the drawings.

[0010] III. Summary of the Invention

[0011] In summary, the data provided by the inventors of this invention demonstrate that Epstein-Barr virus-like particles (EB-VLPs) not only contain most of the viral glycoproteins but also induce numerous glycoprotein-specific antibodies in immunized rodents. Among these, the inventors discovered antibodies with novel specificity (including BILF2) and identified several antibodies that interfere with viral infection on typical EBV target cells (i.e., B cells and epithelial cells). Although the EBV membrane glycoprotein BILF2 is largely uncharacterized and appears to constitute only a small fraction of the total EBV membrane antigen, the inventors found that immunization of rodents with EB-VLPs produced antibodies with previously uncharacterized specificity (including BILF2). These antibodies are capable of interfering with viral infection, and further characterization of the EBV immune response in humans and rodents revealed that antibodies targeting BILF2 unexpectedly constitute a core element of the adaptive immune response to EBV infection and subsequent immunization with EB-VLPs or other immunization strategies.

[0012] Furthermore, it has been demonstrated that the antibodies provided by this invention compete with antibodies against BILF2 in human serum for the same epitopes, thus demonstrating the preventive and / or therapeutic value of the antibodies of this invention, as demonstrated by Liu et al. (2019), who showed that 94% of healthy EBV-positive individuals possessed IgA antibodies against BILF2. These antibodies were obtained by the inventors of this invention by immunizing rodents with the compositions of this invention described herein.

[0013] Specifically, the inventors immunized rodents with compositions containing BILF2, such as EB-VLPs or extracellular vesicles (EVs) carrying BILF2 on their membranes, and elicited antibodies that reduced or even prevented EBV infectivity in human epithelial cells. This finding is surprising because BILF2 is a more or less uncharacterized EBV protein, and antibodies obtained to date have not shown any potential for preventing non-B cell infection (see Maskett et al., 1990, p. 2549, left column, last paragraph and p. 2549, right column, first paragraph). As stated above, the antibodies provided by this invention compete with antibodies against BILF2 in human serum for the same epitope, demonstrating the fact that the antibodies of this invention will have preventive and / or therapeutic value. In fact, EBV-infected epithelial-derived cancers are well known in the art. All tumor cells of nasopharyngeal carcinoma (NPC) and approximately 10% of gastric cancer cases are infected with EBV, which has been classified by the WHO as a Group 1 human carcinogen capable of inducing various tumors in humans. Therefore, the antibody provided by this invention can interfere with EBV infection of epithelial cells, thereby preventing human cancer.

[0014] In summary, this invention provides an EBV vaccine that can induce a broad humoral immune response that can protect against various EBV-related human diseases. Therefore, antibodies obtained by immunizing rodents with the EBV vaccine of this invention can also protect against various EBV-related human diseases because these antibodies reduce the infectivity of EBV to human epithelial cells. Another conclusion is that vaccines based solely on BILF2 can induce an immune response that interferes with EBV infection of epithelial cells and thus prevent EBV-related diseases in humans. Furthermore, vaccines based on BILF2 in combination with other EBV (glycoproteins) can also induce an immune response that interferes with EBV infection of epithelial cells and B cells.

[0015] Therefore, the present invention relates to a composition comprising

[0016] a) BILF2 glycoprotein of Epstein-Barr virus (EBV),

[0017] b) The nucleic acid encoding the BILF2 glycoprotein of EBV, or

[0018] c) Extracellular vesicles (EVs) or EBV-like particles (VLPs) displaying the BILF2 glycoprotein of EBV on their surface.

[0019] In a method of using the composition to induce an antibody against the BILF2 glycoprotein, the antibody reduces the infectivity of EBV to human epithelial cells.

[0020] Preferably, in the composition, the antibody prevents EBV infection of human epithelial cells.

[0021] Preferably, in the composition, the BILF2 glycoprotein is produced by and isolated from mammalian cells.

[0022] Preferably, in the composition, the nucleic acid is DNA or RNA, and preferably, the DNA is contained in a vector.

[0023] Preferably, the composition comprises

[0024] a) RNA encoding the BILF2 glycoprotein of EBV, or

[0025] b) Extracellular vesicles (EVs) or EBV-like particles (VLPs) displaying the BILF2 glycoprotein of EBV on their surface.

[0026] In a method of using the composition to induce an antibody against the BILF2 glycoprotein, the antibody reduces the infectivity of EBV to human epithelial cells.

[0027] Preferably, in the composition, the EV or VLP expresses BILF2, which is expressed alone or in combination with one or more other EBV glycoproteins.

[0028] Preferably, in the composition, the other EBV glycoproteins may include gp350, gB, gp42, gH, gL, gM, gN, BMRF2, BDLF2, BDLF3, BILF1, and BARF1.

[0029] Preferably, in the composition, the VLP further comprises one or more EBV structural proteins.

[0030] Preferably, in the composition, the one or more EBV structural proteins include fusion proteins (F), matrix proteins (M) (also known as membranes), or nucleocapsid proteins (N).

[0031] Preferably, the composition further comprises one or more pharmaceutically acceptable carriers.

[0032] Preferably, the composition further comprises one or more adjuvants.

[0033] Preferably, the composition is used in a method of treating or preventing disease.

[0034] Preferably, the composition is used in a method of treating or preventing a disease, wherein the disease is cancer or an infection, wherein the cancer is preferably an epithelial cancer, and the infection is preferably EBV infection, most preferably EBV infection of epithelial cells.

[0035] Preferably, the composition is used in a method of treating or preventing a disease, wherein the disease is cancer, and the cancer is selected from medulloblastoma, retinoblastoma, Burkitt lymphoma, Hodgkin lymphoma, oral cancer, skin cancer, basal cell carcinoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, gastric cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma, bladder cancer, anal cancer, chondrosarcoma, osteosarcoma, sarcoma, primitive neuroectodermal carcinoma (primitive neuroectodermal tumor (PNET)) and combinations thereof.

[0036] The present invention further provides an antibody for the BILF2 glycoprotein against EBV, wherein the antibody binds to epitopes within amino acids 41-160 of SEQ ID NO: 25 and is capable of reducing the infectivity of EBV to human epithelial cells. Such antibodies can be obtained by (or through) immunization (e.g., of rodents) with the compositions of the present invention. The present invention further provides an antibody for a method of passive immunization against EBV, and / or an antibody for a method of reducing the infectivity of EBV to human epithelial cells, and / or an antibody for a method of preventing EBV infection of human epithelial cells.

[0037] Preferably, the antibody of the present invention binds to epitopes in amino acids 80 to 160 or 129 to 143 of SEQ ID NO: 25 and is able to reduce the infectivity of EBV to human epithelial cells.

[0038] Preferably, the antibody is

[0039] (a) An antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence having at least 80%, 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region having an amino acid sequence having at least 80%, 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 8.

[0040] (b) An antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence having at least 80%, 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region having an amino acid sequence having at least 80%, 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 16.

[0041] (c) An antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 23, and said light chain variable region having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 24; or

[0042] (d) An antibody that binds to the same epitope as any of the antibodies in (a) to (c) of the EBV BILF2 protein shown in SEQ ID NO: 25.

[0043] More preferably, the antibody of the present invention is

[0044] (a) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 3, and

[0045] The light chain variable region comprises a light chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 6;

[0046] (b) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 9, a heavy chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 10, and a heavy chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 11, and

[0047] The light chain variable region comprises a light chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 12, a light chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 13, and a light chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 14;

[0048] (c) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 17, a heavy chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 18, and a heavy chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 19, and

[0049] A light chain variable region comprising a light chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 20, a light chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 21, and a light chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 22; or

[0050] (d) An antibody that binds to the same epitope as any of the antibodies in (a) to (c) of the EBV BILF2 protein shown in SEQ ID NO: 25.

[0051] More preferably, the antibody of the present invention is

[0052] (a) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and

[0053] The light chain variable region comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0054] (b) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11, and

[0055] The light chain variable region comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 14.

[0056] (c) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19, and

[0057] A light chain variable region comprising light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 20, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 21, and light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 22; or

[0058] (d) An antibody that binds to the same epitope as any of the antibodies in (a) to (c) of the EBV BILF2 protein shown in SEQ ID NO: 25.

[0059] Preferably, for the antibody of the present invention, the epitope is determined by PepSpot analysis, amino acid substitution analysis, and / or deletion analysis of the extracellular domain of BILF2 shown in SEQ ID NO: 25. The extracellular domain of BILF2 covers amino acids 18 to 212 of SEQ ID NO: 25.

[0060] Preferably, the antibody is used in a method of passive immunization against EBV.

[0061] Preferably, the antibody is used in a method for reducing the infectivity of EBV to human epithelial cells.

[0062] Preferably, the antibody is used in a method for preventing EBV infection of human epithelial cells.

[0063] The present invention further includes a method for diagnosing whether a human subject has antibodies that reduce EBV infectivity in human epithelial cells, the method comprising determining whether antibodies in a serum sample obtained from the subject competitively bind to any of the aforementioned antibodies to an epitope.

[0064] IV. Detailed Implementation

[0065] The compositions of the present invention comprise the BILF2 glycoprotein of Epstein-Barr virus (EBV), nucleic acid encoding the BILF2 glycoprotein of EBV, or extracellular vesicles (EVs) or EBV-like particles (VLPs) displaying the BILF2 glycoprotein of EBV on their surface, for a method of inducing an antibody against the BILF2 glycoprotein, said antibody reducing the infectivity of EBV to human epithelial cells.

[0066] Preferably, these antibodies prevent EBV infection of human epithelial cells.

[0067] Preferably, the antibody triggered by the composition of the present invention is an antibody as defined herein, such as a neutral antibody that binds to epitopes of BILF2 within 40 to 160, preferably 81 to 160 or 129 to 143 amino acids.

[0068] For the purpose of inducing such antibodies, the compositions of the present invention are administered to humans (subjects) using means and methods known in the art. Administration of the compositions can be achieved through various routes, including but not limited to parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration. Delivery methods are not limited to those described above, and any means for intracellular delivery can be used. Other intracellular delivery methods contemplated by the methods of the present invention include, but are not limited to, liposome encapsulation, nanoparticles, etc. Similarly, the antibodies of the present invention can also be administered to humans (subjects) via the foregoing routes or methods.

[0069] The persons (subjects) mentioned herein may "need" to be administered the compositions and / or antibodies of the present invention. Subjects who "need" to be administered the compositions or antibodies of the present invention are in need of reducing the infectivity of EBV to human epithelial cells or preventing human epithelial cell infection. Such subjects include subjects who exhibit one or more symptoms of the disease described herein and / or are at risk of exhibiting one or more symptoms of the disease described herein.

[0070] Therefore, administering the compositions or antibodies of the present invention to subjects in need includes prophylactic administration of the compositions (i.e., before the disease and / or one or more symptoms of the disease are detectable) and / or therapeutic administration of the compositions (i.e., after the disease and / or one or more symptoms of the disease are detectable). The compositions and antibodies of the present invention can also be used in subjects “at risk of disease.” The term “at risk of…” means that a subject is predisposed to developing and / or exhibiting one or more symptoms of a disease. This predisposition may be genetic (e.g., a specific genetic predisposition to exhibiting one or more symptoms of the disease (such as a hereditary condition)) or due to other factors (e.g., environmental conditions, exposure to harmful compounds present in the environment, including carcinogens, etc.). Subjects “at risk of…” include subjects “having the disease,” i.e., subjects experiencing one or more symptoms of the disease. The present invention is not intended to be limited to any particular sign or symptom. Therefore, the present invention is intended to cover subjects experiencing any range of diseases from subclinical symptoms to full onset, wherein the subjects exhibit at least one sign (e.g., signs and symptoms) associated with the disease.

[0071] The compositions of the present invention that induce the antibodies described herein are preferably used as vaccines, such as preventive or therapeutic vaccines.

[0072] The preventive or therapeutic vaccines described in this article all induce antibodies against EBV BILF2, which reduce the infectivity of EBV to human epithelial cells.

[0073] Vaccines are typically used for active immunization. In the context of this invention, the compositions described herein induce antibodies against EBVBILF2, which reduce the infectivity of EBV to human epithelial cells.

[0074] Vaccines increase a subject's immunity against EBV infection. Therefore, such compositions are used as "EBV vaccines" and can refer to therapies that increase a subject's immunity against EBV. Thus, in specific embodiments, vaccines can be administered prophylactically, for example, to subjects in an immunologically naive state (e.g., subjects who have not previously been exposed to EBV). In specific embodiments, vaccines can be administered therapeutically to subjects who have already been exposed to EBV. Therefore, vaccines can be used to improve symptoms associated with EBV infection, such as lymphoproliferative disorders. Vaccines can also reduce the risk of cancers (e.g., nasopharyngeal carcinoma and / or gastric cancer) or smooth muscle tumors.

[0075] In specific implementation plans, preventative vaccines can reduce, control, or eliminate primary EBV infection.

[0076] In specific implementations, therapeutic vaccines can reduce, control, or eliminate EBV reactivation. In specific implementations, the reduction in EBV reactivation can be determined by measuring the expression of EBV cleavage genes. That is, detecting fewer cells with latent or cleavage EBV genes or detecting lower levels of EBV gene expression can indicate a reduction in EBV load (fewer cells latently infected with EBV) and reactivation (fewer cells supporting de novo viral production).

[0077] Those skilled in the art will understand that the immune system is generally capable of producing innate and adaptive immune responses. Typically, innate immune responses are characterized by being largely non-antigen-specific and / or failing to produce immune memory. Adaptive immune responses are characterized by being largely antigen-specific, maturing over time (e.g., increasing affinity and / or cohesion to antigens), and generally producing immune memory. While these and other functional differences between innate and adaptive immunity can be discerned, those skilled in the art will understand that the innate and adaptive immune systems can integrate and therefore work synergistically.

[0078] EBV infection of epithelial target cells is a multi-step process. In the first step, EBV binds to target cells via various host cell surface receptors and multiple viral envelope glycoproteins. In some cases, EBV binding can induce activation of signaling pathways. After binding to cell surface receptors, the viral membrane fuses with the host membrane (either through direct membrane fusion or fusion with the endosomal membrane). The viral capsid is then transported to the perinuclear region in the cytosol. Once it reaches the nuclear pores, the viral genome is released into the nucleus through the pores.

[0079] In the context of this invention, the “infectivity” of EBV refers to its ability to invade cells, particularly human epithelial cells. Therefore, infectivity leads to infection and subsequent disease in humans. Thus, for the purposes of this invention, the term “infectivity” includes infection. That is, without being bound by theory, infection as a result of infectivity is a two-step process. After infection, (i) EBV enters an initial phase, after which it remains latent in infected cells for several days, possibly weeks or months. Upon reactivation, (ii) the latently infected cells become viral factories, thereby causing biochemical exhaustion and cell death. In other words, EBV has a biphasic life cycle: latent infection – breakdown – lytic reactivation and viral release, thereby producing progeny. This is a characteristic of EBV that is unknown in any other viral species.

[0080] As used herein, the term "infection" refers to the invasion of EBV into a host. EBV is considered "infectious" when it is able to invade and replicate or multiply in a human. "EBV infection" specifically refers to the invasion of EBV into a human, such as human cells and tissues. As described herein, EBV invades humans through epithelial cells, and the compositions of the present invention can induce an antibody against BILF2, which reduces the infectivity of EBV to human epithelial cells. Furthermore, the antibodies described herein reduce the infectivity of EBV to human epithelial cells.

[0081] As used herein, “reduction of human epithelial cell infectivity” can also be used as the term “neutralization of human epithelial cell infectivity.” This term refers to / is the property of antibodies induced by the compositions described herein. Therefore, the compositions described herein induce neutralizing antibodies. As used herein, the term “inducing antibody” is defined as an antibody that induces EBV BILF2.

[0082] The term "reduction or neutralization of human epithelial cell infectivity" refers to the percentage reduction in EBV infectivity of human epithelial cells in the presence of antibodies, compared to the EBV infectivity in the absence of antibodies. For example, if the number of EBV-infected cells in a sample is half that in the absence of antibodies, this can be calculated as 50% neutralization. In specific embodiments, "neutralization or reduction of human epithelial cell infectivity" can refer to at least 40% neutralization (or reduction), at least 50% neutralization (or reduction), at least 60% neutralization (or reduction), at least 70% neutralization (or reduction), at least 80% neutralization (or reduction), 90% neutralization (or reduction), 95% neutralization (or reduction), or even 100% neutralization (or reduction).

[0083] This means that if at least 40% neutralization (or reduction) is present, only 60% of human epithelial cells will be infected with EBV. Similarly, if at least 50% neutralization (or reduction) is present, only 50% of human epithelial cells will be infected with EBV. Likewise, if at least 60% neutralization (or reduction) is present, only 40% of human epithelial cells will be infected with EBV. Moreover, if at least 70% neutralization (or reduction) is present, only 30% of human epithelial cells will be infected with EBV. Furthermore, if at least 80% neutralization (or reduction) is present, only 20% of human epithelial cells will be infected with EBV; if at least 90% neutralization (or reduction) is present, only 10% of human epithelial cells will be infected with EBV; if at least 95% neutralization (or reduction) is present, only 5% of human epithelial cells will be infected with EBV; or if 100% neutralization (or reduction) is present, no human epithelial cells will be infected with EBV. Therefore, the higher the degree of neutralization (or reduction), the fewer epithelial cells a person infected with EBV has.

[0084] However, ideally, the neutralization (or reduction) enables the antibodies described herein to prevent EBV infection of human epithelial cells.

[0085] As the epithelial cell, any human epithelial cell can be used, including human epithelial cell lines. HEK293 cells can also be used, as HEK293 is known to be a substitute for human epithelial cells.

[0086] The preferred neutralization assay used in this invention is described in Example 6. In short, HEK293 target cells are used as epithelial cell targets for EBV. The original hybridoma cell culture supernatant or purified monoclonal antibody is incubated with the EBV stock solution. The EBV stock solution encoding GFP contains 1 × 10⁻⁶ oz. 6 Green Raji units (GRUs) were added to HEK293 cells supplemented with fetal bovine serum, mixed, and plated in individual wells of a multi-well cluster plate. Cells were trypsinized, resuspended, and analyzed by flow cytometry and a high-throughput sample unit. Gated GFP-positive cells were used, and the mean percentage (approximately 15%) of GFP-positive cells in controls without hybridoma cell culture supernatant or purified monoclonal antibody was set as 100% for normalization.

[0087] "Epstein-Barr virus," "EBV," "human herpesvirus 4," and "HHV-4" are used interchangeably to refer to oncogenic human herpesviruses. EBV is a cause of diseases such as acute infectious mononucleosis (AIM or IM, also known as glandular fever). It is also associated with certain forms of cancer, such as Hodgkin's lymphoma, Burkitt's lymphoma, nasopharyngeal carcinoma, and conditions associated with human immunodeficiency virus (HIV), such as hairy leukoplakia and central nervous system lymphoma. EBV infects the B cells and epithelial cells of the immune system. Once the initial lytic infection of the virus is controlled, EBV will remain latent in an individual's B cells for the remainder of their life due to its complex life cycle, which includes alternating latent and lytic phases.

[0088] In addition to the aforementioned medical conditions caused by EBV, patients with primary or secondary immunodeficiency (such as transplant recipients) are particularly at risk of developing EBV-related diseases due to the detrimental effects of immunosuppressants on the immune control of EBV-infected B cells. EBV-related post-transplant lymphoproliferative disease (PTLD) is a significant form of post-transplant complication, occurring in up to 20% of organ recipients (Everly et al., 2007; Taylor et al., 2005). Importantly, immunocompromised transplant recipients who are immunologically naive to EBV at the onset of immunosuppression are at particularly high risk of developing life-threatening EBV+ post-transplant lymphoproliferative disease (PTLD) due to primary EBV infection (e.g., often due to the high prevalence of EBV, which is transmitted through donor organs after transplantation). These patients cannot effectively elicit EBV-specific T cells, which play a crucial role in controlling the proliferation of EBV-infected B cells, due to impaired T-cell immunity resulting from exposure to immunosuppressive drugs. In contrast, patients who are EBV seropositive at transplantation have a much lower risk of developing PTLD, demonstrating the crucial role of EBV-specific T cells, which are ready to eliminate virus-infected cells. Typically, patients who are EBV serone before transplantation have a much higher risk of developing EBV-related diseases because transmission of donor EBV into the transplanted organ or natural infection with the virus can lead to lymphoproliferative disorders in EBV serone recipients post-transplantation (e.g., Mendoza et al., 2006; Swerdlow et al., 2000). As with many virus-related diseases, vaccination is a promising approach to preventing and / or treating viral infection and its consequences in the host, and this is also true for reducing the high risk of PTLD in seronegative patients by immunizing them against EBV before transplantation.

[0089] Although EBV can cause acute illness and is believed to be involved in the pathogenesis of life-threatening diseases, effective methods to reduce EBV infectivity have not yet entered clinical application despite decades of in-depth research. The inventors previously disclosed an Epstein-Barr virus vaccine (WO2012 / 025603) comprising virus-like particles obtainable by transfecting human cells with a modified EBV genome that is packaging-deficient, replication-deficient, and unable to transform B cells. This vaccine was prepared for a method of inducing therapeutic or prophylactic EBV-specific CD8+ cellular and humoral immune responses in human individuals. This invention further refines and improves upon this finding by identifying the role of the specific EBV glycoprotein BILF2 in inducing and mediating adaptive antibody-mediated immune responses, and demonstrating that immunization with BILF2 is sufficient to induce a durable adaptive immune response that limits the infectivity of BILF2 in epithelial cells.

[0090] The compositions of the present invention comprise the BILF2 glycoprotein of Epstein-Barr virus (EBV), nucleic acid encoding the BILF2 glycoprotein (gp75 / 55) of EBV, or extracellular vesicles (EVs) or EBV-like particles (VLPs) displaying the BILF2 glycoprotein of EBV on their surface.

[0091] As used in this article, "BILF2" refers to the BILF2 protein derived from EBV. However, the term may also refer to the nucleic acid (containing the nucleotide sequence) encoding the BILF2 protein.

[0092] Typically, proteins or polypeptides (including fragments thereof, preferably bioactive fragments, and peptides generally having fewer than 30 amino acids) comprise one or more amino acids coupled to each other via covalent peptide bonds (thus forming an amino acid chain). The term "polypeptide" as used herein describes a group of molecules composed of more than 30 amino acids. Polypeptides can further form polymers, such as dimers, trimers, and higher oligomers, i.e., composed of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc., can be identical or different. Therefore, the corresponding higher-order structures of such polymers are called homodimers or heterodimers, homotrimers or heterotrimers, etc. An example of a heteropolymer is an antibody molecule, which in its naturally occurring form in the human body consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides / proteins, where such modifications are achieved, for example, through post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. The "polypeptide" mentioned herein can also be chemically modified, for example, by polyethylene glycolation. Such modifications are well known in the art.

[0093] Generally, the term "nucleic acid" is well known to those skilled in the art and includes DNA (such as cDNA) and RNA (such as mRNA). Nucleic acids can be double-stranded or single-stranded, linear or circular. The nucleic acid molecules are preferably contained in a vector, which can preferably be contained in a host cell, for example, for its proliferation.

[0094] BILF2 is a single-pass transmembrane type 1 membrane glycoprotein; therefore, the term "BILF2 glycoprotein" is sometimes used. No published data exist on BILF2 since it was initially described as 'gp78 / 55' in the virion. This protein has a protein backbone of approximately 28 kDa and a large number of N-linked or O-linked glycan components (Mackett et al., 1990). BILF2 was first characterized in 1990 (Mackett et al., 1990), but its role in EBV remains poorly understood. BILF2 is an envelope glycoprotein encoded by a late cleavage gene called BILF2 (from which the glycoprotein derives its name). BILF2-derived peptides have been shown to be recognized on the surface of EBV-infected cells by CD8+ T cells from patients with infectious mononucleosis (IM) (Taylor et al., 2015; Pudney et al., 2005). IgG antibodies against BILF2 were detected in 73% of children with endemic Burkitt lymphoma (BL), a nearly 100% EBV-positive B-cell non-Hodgkin lymphoma (Coghill et al., 2020).

[0095] The preferred BILF2 protein covered by the term "BILF2" has the amino acid sequence shown in SEQ ID NO: 25. However, "BILF2" covers more than just the preferred amino acid sequence shown in SEQ ID NO: 25. "BILF2" may also cover BILF2 variants having at least 60%, 70%, 80%, 90%, or higher identity with the amino acid sequence shown in SEQ ID NO: 25. "BILF2" may also cover fragments of BILF2. Such fragments are at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240 amino acids of the amino acid sequence shown in SEQ ID NO: 25.

[0096] Preferred BILF2 nucleotide sequences are shown in SEQ ID NO: 26 or 27. However, BILF2 nucleotide sequences do not only encompass the preferred nucleotide sequences shown in SEQ ID NO: 26 or 27. BILF2 nucleotide sequences may also encompass BILF2 nucleotide sequence variants having at least 60%, 70%, 80%, 90%, or higher identity with the nucleotide sequences shown in SEQ ID NO: 26 or 27. BILF2 nucleotide sequences may also encompass fragments thereof. Such fragments are at least 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, 600, 630, 660, 690, or 720 nucleotides in length of the nucleotide sequence shown in SEQ ID NO: 26 or 27.

[0097] The term "identity" refers to the relationship between sequences of two or more polypeptides (e.g., antibodies or antigens, such as BILF2) or polynucleotides (nucleotide sequences or nucleic acids) determined by sequence comparison. Identity also refers to the degree of sequence correlation between sequences determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of consistent matches between the smaller of two or more sequences, where vacancy alignment (if present) is handled by a specific mathematical model or computer program (e.g., an "algorithm"). The identity of related antigens or nucleic acids can be readily calculated using known methods. The "percentage of identity (%)" applied to polypeptide or polynucleotide sequences 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 the second sequence after sequence alignment and, if necessary, the introduction of vacancy to achieve the maximum percentage of identity. The methods and computer programs used for alignment are well known in the art. It should be understood that identity depends on the calculation of the percentage of identity, but its value may differ due to vacancy and penalty introduced in the calculation. Generally, as determined by sequence alignment procedures and parameters described herein and known to those skilled in the art, a variant of a particular polynucleotide or polypeptide (e.g., an antigen) has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but less than 100% sequence identity with a particular reference polynucleotide or polypeptide. Such tools for alignment include those in BLAST suites (Stephen F. Altschul et al., (1997), “Gapped BLAST and PSTBLAST: a newgeneration of protein database search programs”, Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981)).

[0098] For example, appropriate alignment of nucleic acid sequences can be provided by the local homology algorithm of Smith and Waterman, (1981), Advances in Applied Mathematics 2:482-489. This algorithm can be applied to amino acid sequences using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6): 6745-6763. An exemplary implementation of this algorithm for determining the percentage of sequence identity is provided by Genetics Computer Group (Madison, Wis.) in its "BestFit" application. The default parameters for this method are described in the Wisconsin SequenceAnalysis Package Program Manual, version 8 (1995), available from Genetics Computer Group, Madison, Wis. In the context of this invention, a preferred method for establishing the identity percentage is to use the MPSRCH package, copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif). From this package, the Smith-Waterman algorithm can be employed, using default parameters in the scoring table (e.g., a gap opening penalty of 12, a gap extension penalty of one, and a gap of six). In the generated data, the "match" value reflects "sequence identity." Other suitable procedures for calculating the identity or similarity percentage between sequences are generally known in the art; for example, another alignment procedure is BLAST used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: Genetic code = standard; Filter = none; Chain = both; Cutoff = 60; Expected = 10; Matrix = BLOSUM62; Description = 50 sequences; Sort by = high score; Database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss protein+Spupdate+PIR.Details of these procedures can be found at: https: / / blast.ncbi.nlm.nih.gov / .

[0099] In this invention, BILF2 can be presented to the immune system on the surface of extracellular vesicles (EVs) or virus-like particles (VLPs). As used herein, the term "extracellular vesicle" refers to a lipid bilayer-bound particle ranging from 20 to 10,000 nanometers, formed by secretion from a cell into the extracellular space. As used herein, the term "extracellular vesicle" can also mean "nanoparticle." Therefore, this invention also includes EVs that are nanoparticles, and vice versa. Thus, in the context of this invention, extracellular vesicles (EVs) or EB virus-like particles (VLPs) exhibiting the BILF2 glycoprotein of EBV on their surface can also be nanoparticles carrying the BILF2 glycoprotein, such as EVs. As used herein, the term "virus-like particle" (or "particle") refers to particulate aggregates of EBV polypeptides and membrane lipids that do not contain EBV DNA and are therefore replication-deficient. VLPs in the context of this invention may be categorized under the term "nanoparticle."

[0100] Preferably, BILF2 is expressed by EV or VLP, and BILF2 is expressed alone or in combination with one or more other EBV glycoproteins. These other EBV glycoproteins may include gp350, gB, gp42, gH, gL, gM, gN, BMRF2, BDLF2, BDLF3, BILF1, and BARF1.

[0101] The VLP may also contain one or more EBV structural proteins. These one or more EBV structural proteins include fusion proteins (F), matrix proteins (M), or nucleocapsid proteins (N).

[0102] The compositions of the present invention, particularly BILF2, when presented via EV or VLP, can also elicit a cellular immune response, including a CD4+ and / or CD8+ T cell response. The term "CD4+ or CD8+ T cell response" refers to a T cell immune response characterized by a high proportion of immunogen-specific CD4+ or CD8+ T cells observed in the total responding T cell population after vaccination. The total immunogen-specific T cell response can be determined, for example, by IFN-γ ELISPOT analysis. The immunogen-specific CD4+ or CD8+ T cell immune response can be determined, for example, by intracellular cytokine staining (ICS) analysis (Horton et al., (2007), J Immuol Methods 323(1): 39-54).

[0103] The phrase "EBV DNA-free" means that the particles do not contain EBV DNA, i.e., EBV DNA was not detected. Specifically, according to the invention, the term "DNA" includes any DNA, such as cDNA or genomic DNA. The term "DNA" as used in this context also includes DNA mimics known in the art, such as synthetic or semi-synthetic derivatives and mixed polymers of DNA, including sense and antisense strands. Such DNA mimics or DNA derivatives of the invention comprise phosphate-thiocyanate nucleic acids. Therefore, the particles must not contain a DNA sequence identical to an EBV DNA sequence, wherein the sequence preferably relates to an EBV gene sequence. Furthermore, the particles must not contain nucleic acid sequences having at least (for each value) 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, and at least 75% sequence identity with wild-type EBV nucleic acid sequences. The degree of sequence identity of the nucleic acid sequences can be calculated by those skilled in the art using methods well known to them, and may include algorithms that automatically perform sequence data alignment and sequence homology calculations. Moreover, the particles must not contain DNA sequences that, when expressed, produce functionally similar peptides to EBV, wherein the functional similarity preferably relates to B-cell transformation capacity. Methods for testing the functional similarity of peptides include computer simulations and in vitro, ex vivo, and in vivo assays well known to those skilled in the art. For example, to determine functional similarity, a deletion mutant of EBV can be generated and complementation analysis performed. Specifically, the deletion mutant of EBV is characterized by the deletion of a sequence of a gene encoding a peptide, for example, essential for B cell transformation. As a result, the deletion mutant cannot transform B cells. To detect the functional similarity between a DNA sequence and a deleted EBV sequence, the test DNA is provided to the deletion mutant, for example, by integrating the test DNA into the genomic DNA of the deletion mutant. Subsequently, it is determined whether the modified deletion mutant can transform B cells, i.e., by complementary deletion and the generation of EBV functionally similar to wild-type EBV. Variations of the complementation analysis are known in the art and can be further tailored to specific needs based on the technical knowledge of those skilled in the art.

[0104] In this invention, EVs and VLPs can be used to expose the immune system to BILF2 and thus to methods for inducing antibodies. In the context of this invention, a "method for inducing antibodies" refers to a protocol or procedure designed to stimulate the immune system to produce specific antibodies against a target antigen in a living organism. By inducing antibody production, and subsequently maintaining the ability to produce said antibodies via the generation of persistent memory B cells upon re-exposure to the relevant antigen, this method is expected to result in adaptive immunity that reduces the infectivity and / or virulence of the relevant pathogen (here, EBV).

[0105] In this invention, BILF2 can also be presented to the immune system via a composition comprising a nucleic acid containing the BILF2 glycoprotein encoding EBV. The nucleic acid may be DNA or RNA. The DNA or RNA has an open reading frame encoding BILF2. In some embodiments, the RNA (e.g., mRNA) further comprises (at least one) 5' UTR, 3' UTR, a polyadenylated tail, and / or a 5' cap.

[0106] Nucleic acids are polymers of nucleotides (nucleotide monomers), also known as polynucleotides. Nucleic acids can be or may include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose conformation, α-LNA with α-L-ribose conformation (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-α-LNA with 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof.

[0107] Messenger RNA (mRNA) is any ribonucleic acid that encodes (at least one) protein (a naturally occurring, non-naturally occurring, or modified polymer of amino acids) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein. In the context of this invention, such a protein is BILF2. Those skilled in the art will understand that, unless otherwise stated, the nucleic acid sequences described herein may be referred to as “T” in representative DNA sequences, but when the sequence represents RNA (e.g., mRNA), “T” is replaced with “U”. Therefore, any DNA disclosed and identified herein by a specific sequence identification number also discloses a corresponding RNA (e.g., mRNA) sequence complementary to said DNA, wherein each “T” in said DNA sequence is replaced with “U”.

[0108] An open reading frame (ORF) is a continuous segment of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and terminates with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). ORFs typically encode proteins. It should be understood that the sequences disclosed herein may further contain other elements, such as 5' and 3' UTRs, but unlike ORFs, these elements are not necessarily required to be present in the vaccines disclosed herein.

[0109] In some embodiments, the nucleic acid encoding BILF2 is codon-optimized, such as the nucleotide sequence shown in SEQ ID NO: 27. Codon optimization methods are well known in the art. In some embodiments, codon optimization can be used to match codon frequencies in the target organism and the host organism to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base strings that may impair gene construction or expression; customize transcription and translation control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; add, remove, or reorganize protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; regulate translation rates to allow proper folding of various protein domains; or reduce or eliminate problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In some embodiments, optimization algorithms are used to optimize open reading frame (ORF) sequences.

[0110] In some embodiments, the codon-optimized sequence has less than 95% sequence identity with a naturally occurring or wild-type sequence ORF (e.g., a naturally occurring or wild-type mRNA sequence encoding BILF2). In some embodiments, the codon-optimized sequence has less than 90% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding BILF2). In some embodiments, the codon-optimized sequence has less than 85% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding BILF2). In some embodiments, the codon-optimized sequence has less than 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding BILF2). In some embodiments, the codon-optimized sequence has less than 75% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding BILF2).

[0111] In some embodiments, the codon-optimized sequence has 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding BILF2). In some embodiments, the codon-optimized sequence has 65% to 75% or about 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding BILF2).

[0112] In some implementations, the immunogenicity of BILF2 encoded by codon-optimized sequences is the same as, or higher than, that of BILF2 encoded by non-codon-optimized sequences (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% or higher).

[0113] When transfected into mammalian host cells, the modified mRNA exhibits stability for 12–18 hours, or longer than 18 hours, such as 24, 36, 48, 60, 72, or longer, and is capable of being expressed by mammalian host cells.

[0114] In some implementations, codon-optimized RNA can be RNA with enhanced G / C levels. The G / C content of nucleic acid molecules (e.g., mRNA) can affect RNA stability. RNA with increased amounts of guanine (G) and / or cytosine (C) residues may be functionally more stable than RNA containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides.

[0115] As described herein, the nucleic acid encoding BILF2 is DNA that can be contained in a vector. The vector can be a circular plasmid or a linear nucleic acid. Circular plasmids and linear nucleic acids are capable of directing the expression of a specific nucleotide sequence (in the context of this invention, the nucleic acid contained in the vector is DNA) in suitable cells. Vectors containing nucleic acid constructs can be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components.

[0116] In this invention, the method of inducing antibodies against BILF2 reduces the infectivity of EBV to human epithelial cells. This is highly relevant to the spread and initial infection of EBV in humans, which typically occurs via saliva transmission, leading to infection of oropharyngeal epithelial cells, which can subsequently result in B-cell infection (Houene et al., 2021).

[0117] In addition to the compositions described above, the compositions according to the invention may also contain pharmaceutically acceptable carriers, including any carriers that do not induce an immune response or any other adverse reaction that is harmful to the individual receiving the composition.

[0118] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refer to solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents that are compatible with drug administration. The use of such media and reagents for pharmaceutically active substances is well known in the art. In some embodiments, the pharmaceutically acceptable carrier or excipient is not naturally occurring.

[0119] Suitable carriers are typically large, slowly metabolizing macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Other suitable pharmaceutically acceptable carriers are well known in the art. Additionally, these carriers can also act as other immunostimulants, which will be described in more detail below. Furthermore, the composition may contain diluents such as water, saline, glycerol, ethanol, etc. Furthermore, the vaccine may contain substances necessary for formulation purposes, such as emulsifiers and / or pH buffers. Any combination of the above substances may be incorporated into the compositions of this invention as needed.

[0120] In some embodiments of the invention, the composition is administered in conjunction with one or more adjuvants. As used herein, "adjuvant" means a substance that itself increases or modulates the immune response of a subject. Adjuvants are commonly used to modify or enhance the effectiveness of vaccines by stimulating the immune system to respond more strongly, thereby providing enhanced immunity against a specific disease. Some adjuvants accomplish this task by mimicking a specific set of evolutionarily conserved molecules (i.e., so-called pathogen-associated molecular patterns), including liposomes, lipopolysaccharides, molecular cages of antigens, components of bacterial cell walls, and endocytosed nucleic acids (e.g., RNA, double-stranded RNA, single-stranded DNA, and DNA containing unmethylated CpG dinucleotides). In this context, adjuvants are used to enhance the immune response to plasmids and / or EBV vectors described herein and in the context of this invention.

[0121] In some embodiments of the present invention, in a method of using the composition described herein for treating or preventing a disease, preferably, the disease is cancer or an infection, the cancer is preferably an epithelial-derived cancer, the infection is preferably EBV infection, and most preferably EBV infection of epithelial cells.

[0122] Preferably, the composition described herein is used in a method of treating or preventing a disease, wherein the disease is cancer, and the cancer is selected from: medulloblastoma, retinoblastoma, Burkitt lymphoma, Hodgkin lymphoma, oral cancer, skin cancer, basal cell carcinoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma, bladder cancer, anal cancer, chondrosarcoma, osteosarcoma, sarcoma, primitive neuroectodermal carcinoma (primitive neuroectodermal tumor (PNET)) and combinations thereof.

[0123] When used in the context of a disease or condition, the term “prevention” refers to the preventive application of a composition that prevents or otherwise delays the onset of pathological signs or symptoms of a disease or condition.

[0124] When used in the context of a disease or disease condition, the term “treatment” refers to the relief, improvement, or cure of symptoms of a disease, symptom, or condition, or it can refer to completely or partially halting the biochemical basis of a disease at the molecular level, such as stopping viral replication.

[0125] The present invention further provides an antibody against the BILF2 glycoprotein of EBV, wherein the antibody binds to epitopes within amino acids 41 to 160 of SEQ ID NO: 25 and is capable of reducing the infectivity of EBV to human epithelial cells. Such antibodies can be obtained by (or through) immunization with the compositions of the present invention (e.g., rodents); see also Examples 4 to 6. The present invention further provides an antibody for use in methods of passive immunization against EBV, and / or for reducing the infectivity of EBV to human epithelial cells, and / or for preventing EBV infection of human epithelial cells.

[0126] Preferably, the antibody binds to epitopes in amino acids 80 to 160 or 129 to 143 of SEQ ID NO: 25 and is capable of reducing the infectivity of EBV to human epithelial cells.

[0127] Preferably, for the antibody of the present invention, the epitope is determined by PepSpot analysis and deletion analysis of the extracellular domain of BILF2 shown in SEQ ID NO: 25. The extracellular domain of BILF2 covers amino acids 18 to 212 of SEQ ID NO: 25.

[0128] An epitope encompasses any determinant cluster capable of being bound by an antibody. An epitope is a region of a polypeptide that is bound by an antibody targeting that region. Such polypeptide regions include specific residues that directly contact the antibody. These specific residues include amino acids, sugar chains linked to amino acids, or both. In a specific embodiment, an epitope refers to a binding site of the antibody on the polypeptide. Antibodies bind linear epitopes (e.g., epitopes comprising a segment of 5 to 15 consecutive amino acids), or antibodies bind to a three-dimensional structure formed by the spatial arrangement of several short segments of a polypeptide target. A three-dimensional epitope recognized by an antibody (e.g., through an epitope recognition site or complementary site) can be considered a three-dimensional surface feature of the epitope molecule. These features precisely adapt to the corresponding binding site of the antibody, thereby facilitating binding between the antibody and its target polypeptide. In a specific embodiment, an epitope can be considered to have two layers: (i) a “covered patch,” which can be viewed as a shadow that the antibody will project; and (ii) individual participating side chain and backbone residues. Binding is then a combination of ionic interactions, hydrogen bonds, and hydrophobic interactions.

[0129] To define the epitope targeted by the antibody, the epitope must be identified. Appropriate methods for defining the epitope include PepSpot analysis and / or deletion analysis.

[0130] Peptide scanning (PepSpot analysis) is commonly used to locate linear epitopes in polypeptide antigens. The primary sequence of a polypeptide is synthesized sequentially on activated cellulose, with peptides overlapping each other. Antibodies, whose ability to detect or recognize specific antigens / epitaxies is to be tested, recognize some peptides by conventional colorimetric methods (e.g., using a secondary antibody with horseradish peroxidase and 4-chloronaphthol and hydrogen peroxide), chemiluminescence reactions, or similar methods known in the art. In the case of chemiluminescence reactions, the reaction can be quantified. If the antibody reacts with a set of overlapping peptides, the minimum sequence of amino acids required for the reaction can be deduced; see Illustrative Example 5 and Table 2.

[0131] The same analysis can reveal two reactive peptide clusters that are far apart, indicating the recognition of discontinuous epitopes, i.e. conformational epitopes, in antigenic peptides.

[0132] Deletion analysis is performed by separately deleting portions of the peptide. The resulting deletion variants are then tested to determine whether the antibody binds to them. In the absence of binding, the missing amino acid may be part of an epitope.

[0133] Preferably, the antibody of the present invention is

[0134] (a) An antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence having at least 80%, 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region having an amino acid sequence having at least 80%, 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 8.

[0135] (b) An antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence having at least 80%, 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region having an amino acid sequence having at least 80%, 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 16.

[0136] (c) An antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 23, and said light chain variable region having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 24; or

[0137] (d) An antibody that binds to the same epitope as any of the antibodies in (a) to (c) of the EBV BILF2 protein shown in SEQ ID NO: 25.

[0138] Preferably, the antibody of the present invention is

[0139] (a) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 3, and

[0140] The light chain variable region comprises a light chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 6;

[0141] (b) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 9, a heavy chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 10, and a heavy chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 11, and

[0142] The light chain variable region comprises a light chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 12, a light chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 13, and a light chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 14;

[0143] (c) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 17, a heavy chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 18, and a heavy chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 19, and

[0144] A light chain variable region comprising a light chain CDR1 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 20, a light chain CDR2 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 21, and a light chain CDR3 having an amino acid sequence having at least 80%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID NO: 22; or

[0145] (d) An antibody that binds to the same epitope as any of the antibodies in (a) to (c) of the EBV BILF2 protein shown in SEQ ID NO: 25.

[0146] Preferably, the antibody of the present invention is

[0147] (a) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and

[0148] The light chain variable region comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0149] (b) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11, and

[0150] The light chain variable region comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 14.

[0151] (c) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19, and

[0152] A light chain variable region comprising light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 20, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 21, and light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 22; or

[0153] (d) An antibody that binds to the same epitope as any of the antibodies in (a) to (c) of the EBV BILF2 protein shown in SEQ ID NO: 25.

[0154] When two antibodies recognize the same or spatially overlapping epitopes, the antibody binds to the epitope that the reference antibody (e.g., an antibody as defined herein in sections (a) through (c)) binds to is “the same epitope”. The most widely used and rapid method for determining whether two epitopes bind to the same or spatially overlapping epitopes is competitive analysis, which can be configured in a variety of different ways, using labeled antigens or labeled antibodies.

[0155] Amino acid sequence modifications of the antibodies described herein have been considered. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibodies of the present invention are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding such antibodies or by peptide synthesis.

[0156] Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct possesses the desired characteristics. Amino acid alterations can also modify the post-translational process of the antibody, such as changing the number or position of glycosylation sites. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in the CDR may be substituted, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids in the frame region (FR) may be substituted. Substitutions are preferably conserved substitutions. Alternatively, 1, 2, 3, 4, 5, or 6 amino acids may be inserted or omitted in each CDR (depending on the length of the CDR, of course), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be inserted or omitted in each FR.

[0157] Another type of variant is the amino acid substitution variant. These variants preferably have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in the antibody replaced by different residues. The sites of most interest in substitution mutagenesis include the CDR of the heavy chain and / or light chain, especially the hypervariable region, but FR changes in the heavy chain and / or light chain are also considered.

[0158] For example, if the CDR sequence contains 6 amino acids, it is conceivable that one, two, or three of these amino acids are substituted. Similarly, if the CDR sequence contains 15 amino acids, it is conceivable that one, two, three, four, five, or six of these amino acids are substituted.

[0159] Generally, if amino acid substitutions are present in one or all of the CDRs in the heavy chain and / or light chain, it is preferred that the resulting "substituted" sequence be at least 80%, 90%, or 95% identical to the "original" CDR sequence. This means that the degree of identity with the "substituted" sequence depends on the length of the CDR. For example, a CDR with 5 amino acids preferably has 80% identity with its substituted sequence in order to have at least one amino acid substitution. Therefore, antibody CDRs have different degrees of identity with their substituted sequences; for example, CDRL1 may have 80%, while CDRL3 may have 90%.

[0160] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution is envisioned as long as the antibody retains its ability to bind to BILF2 and / or its CDR has identity with the subsequently substituted sequence (at least 80%, 85%, or 90% identity with the "original" CDR sequence).

[0161] The substantial modification of the biological properties of the antibodies of this invention is achieved by selecting substitutions that are significantly different in maintaining the following effects: (a) the structure of the polypeptide backbone in the substitution region, for example, sheet or helical conformation; (b) the charge or hydrophobicity of the target site molecule; or (c) the volume of the side chain. Naturally occurring residues are categorized into the following groups based on common side chain properties: (1) hydrophobic: leucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; (3) acidic: Asp, Glu; (4) basic: Asn, Gln, His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0162] Non-conservative substitutions may require exchanging members of one of these categories for another. Any cysteine ​​residue that does not participate in maintaining the correct conformation of the antibody can be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent undesirable cross-linking. Conversely, cysteine ​​bonds can be added to the antibody to enhance its stability.

[0163] A particularly preferred type of substitution variant involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further development exhibit improved biological properties relative to the parent antibody that produced them. A convenient method for generating such substitution variants involves phage-displayed affinity maturation. Briefly, several hypervariable sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The resulting antibody variants are displayed monovalently on filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Alternatively, or additionally, analysis of the crystal structure of the antigen-antibody complex to identify contact points between the antibody and BILF2 may be beneficial. According to the techniques described herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, the group of variants is screened as described herein, and antibodies that exhibit superior properties in one or more relevant analyses can be selected for further development.

[0164] This article considers other modifications to antibodies. For example, antibodies can be linked to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyethylene, or copolymers of polyethylene glycol and polypropylene glycol. Antibodies can also be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by cohesive techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in crude emulsions.

[0165] The term "antibody" is used herein in its broadest sense, in various grammatical forms, and specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, polymers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, TCAs, single-chain Fv (scFv), nanobodies, and also includes antibody fragments such as Fab or F(ab)2, provided they exhibit the desired biological activity. Antibodies can be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or antibodies derived from other species. The antibodies of this invention are preferably monoclonal antibodies.

[0166] The term antibody can refer to a full-length heavy chain, full-length light chain, or complete immunoglobulin molecule; or an immunoactive portion of any of these polypeptides, i.e., a polypeptide containing an antigen-binding site that specifically binds to a target antigen or a portion thereof, said targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules, including engineered subclasses with altered Fc moieties that provide reduced or enhanced effector cell activity. The light chain of the subject antibody can be a κ light chain (Vκ) or a λ light chain (Vλ). Immunoglobulins can be derived from any species.

[0167] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical, except for the possible presence of naturally occurring mutations, which may be present in trace amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The monoclonal antibodies of this invention can be prepared by hybridoma methods or via recombinant protein production methods.

[0168] In the context of antibodies, the term "variability" refers to the fact that certain portions of the antibody's variable domain differ widely in sequence between antibodies and are responsible for the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. It is concentrated in three segments called hypervariable regions in the variable domains of the light and heavy chains. The more conserved portions of the variable domain are called frame regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, predominantly adopting a [3-sheet configuration], connected by three hypervariable regions that form loops connecting P-sheets and, in some cases, form part of a P-sheet. The hypervariable regions in each chain are held together closely by the FRs and, together with hypervariable regions from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains do not directly participate in antibody-antigen binding, but exhibit various effector functions, such as antibody participation in antibody-dependent cytotoxicity (ADCC).

[0169] When used herein, the term “hypervariant region” refers to the amino acid residues in an antibody responsible for antigen binding. Hypervariant regions typically contain amino acid residues from the “complementarity-determining region” or “CDR” (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the “hypervariant loop” residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, “CDR” refers to the complementarity-determining region of the antibody. "Frame region" or "FR" residues are variable domain residues other than the hypervariable region / CDR residues defined in this paper.

[0170] The antibody residues in this article are numbered according to the Kabat numbering system and the EU numbering system. When referring to residues in the variable domain (approximately residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is generally used (e.g., the EU index reported by Kabat et al., see above). “EU index in Kabat” refers to the residue number of the human IgG EU antibody. Unless otherwise stated herein, residue numbers in the antibody variable domain refer to residue numbers in the Kabat numbering system. Unless otherwise stated herein, residue numbers in the antibody constant domain refer to residues numbered according to the EU numbering system.

[0171] Preferably, the antibodies of the present invention, or antibodies induced by the compositions of the present invention, are “neutralizing antibodies.” Neutralizing antibodies are antibodies that, upon binding to an epitope within their BILF2 (e.g., an epitope described herein), reduce the infectivity of EBV to human epithelial cells; preferably, these antibodies prevent EBV infection of human epithelial cells. Neutralizing antibodies can also be used as protein therapeutic agents to prevent or treat viral infections. Neutralizing antibodies against EBV typically function by blocking viral entry into cells. To enter cells, EBV first attaches to the cell surface through interactions between proteins on the viral surface and receptor-binding sites on cell surface proteins. Following this attachment, the viral membrane can fuse with the cell membrane, allowing the viral contents to be inserted into the cell. Viral fusion also occurs through interactions between viral proteins and epitopes on cellular protein antigens. The interactions leading to viral attachment to cells and the interactions leading to viral fusion to cells are different, each involving different viral proteins and different cellular proteins. Therefore, neutralizing antibodies can block EBV entry into cells by preventing the virus / cell protein interactions that lead to attachment and / or fusion.

[0172] The antibody against BILF2 of the present invention preferably interacts (or binds) specifically to BILF2. As used herein, the terms "specific interaction," "specifically binding," or "specifically bind(s)" refer to binding domains that exhibit significant affinity for BILF2 and generally do not show significant reactivity to proteins or antigens other than BILF2. "Significant affinity" includes a binding domain with an affinity of about 10... -6 M (KD) or stronger affinity binding. Preferably, when the binding affinity is about 10... -9 Up to 10 -6 M, preferred 10 -9 Up to 10 -7 M or 10 -9 Up to 10 -8 At point M, binding is considered specific; see also Table 2. Therefore, the antibody of the present invention preferably has about 10 -11 Up to 10 -10 M, approximately 10 -10 Up to 10 -9 M, approximately 10 -9 Up to 10 -8 M or 10 -8 Up to 10 -7The KD of M. In particular, by comparing the reaction of the antibody with the target protein or antigen and the reaction of the antibody with proteins or antigens other than BILF2, it is easy to test whether the antibody specifically reacts with or binds to BILF2. Preferably, the antibodies of the present invention substantially do not bind or cannot bind proteins or antigens other than BILF2. The terms "substantially do not bind" or "cannot bind" mean that the antibodies of the present invention do not bind to any other protein or antigen besides BILF2, i.e., do not show a reactivity of more than 30%, preferably no more than 20%, more preferably no more than 10%, particularly preferably no more than 9%, 8%, 7%, 6%, or 5% to proteins or antigens other than BILF2, wherein the binding to BILF2 is set to 100%.

[0173] The antibodies described in this article can passively transfer immunity, thereby protecting human epithelial cells from EBV infection.

[0174] The antibody can be further used in methods of passive immunization against EBV.

[0175] Preferably, the antibody is used in a method of passive immunization against EBV.

[0176] Preferably, the antibody is used in a method for reducing the infectivity of EBV to human epithelial cells.

[0177] Preferably, the antibody is used in a method for preventing EBV infection of human epithelial cells.

[0178] The present invention further provides a method for diagnosing whether a human subject has antibodies that reduce EBV infectivity in human epithelial cells, the method comprising determining whether an antibody in a serum sample obtained from the subject binds to an epitope competing with any antibody described herein (particularly an antibody that binds to an epitope within amino acids 41 to 160 of BILF2, preferably within amino acids 80 to 160 or 129 to 143 of BILF2).

[0179] ***

[0180] This invention can also be summarized as follows:

[0181] (1) A composition comprising

[0182] a) BILF2 glycoprotein of Epstein-Barr virus (EBV),

[0183] b) The nucleic acid encoding the BILF2 glycoprotein of EBV, or

[0184] c) Extracellular vesicles (EVs) or EBV-like particles (VLPs) displaying the BILF2 glycoprotein of EBV on their surface.

[0185] In a method of using the composition to induce an antibody against the BILF2 glycoprotein, the antibody reduces the infectivity of EBV to human epithelial cells.

[0186] (2) The composition for the purpose described in Item 1, wherein the antibody prevents EBV infection of human epithelial cells.

[0187] (3) The composition of any one of the preceding items, wherein the BILF2 glycoprotein is produced by and isolated from mammalian cells.

[0188] (4) The composition described in item 1 or 2, wherein the nucleic acid is DNA or RNA.

[0189] (5) The composition for the use described in Item 4, wherein the DNA is contained in a vector.

[0190] (6) The composition of Project 1, wherein the EV or VLP expresses BILF2, and the BILF2 is expressed alone or in combination with one or more other EBV glycoproteins.

[0191] (7) The composition described in item 6, wherein the other EBV glycoproteins may include gp350, gB, gp42, gH, gL, gM, gN, BMRF2, BDLF2, BDLF3, BILF1 and BARF1.

[0192] (8) The composition described in item 6 or 7, wherein the VLP further comprises one or more EBV structural proteins.

[0193] (9) The composition of any one of items 6 to 8, wherein the one or more EBV structural proteins include a fusion protein (F), a matrix protein (M), or a nucleocapsid protein (N).

[0194] (10) The composition of any one of the preceding items further comprises one or more pharmaceutically acceptable carriers.

[0195] (11) The composition for any of the uses described in the preceding items further comprises one or more adjuvants.

[0196] (12) The composition of any one of items 1 to 11, in a method of treating or preventing disease.

[0197] (13) The composition of item 12, wherein the disease is cancer or infection, wherein the cancer is preferably an epithelial cancer, and the infection is preferably EBV infection, most preferably EBV infection of epithelial cells.

[0198] (14) The composition described in item 12 or 13, wherein the disease is cancer, and the cancer is selected from: medulloblastoma, retinoblastoma, Burkitt lymphoma, Hodgkin lymphoma, oral cancer, skin cancer, basal cell carcinoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, gastric cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma, bladder cancer, anal cancer, chondrosarcoma, osteosarcoma, sarcoma, primitive neuroectodermal carcinoma (primitive neuroectodermal tumor (PNET)) and combinations thereof.

[0199] (15) An antibody against the BILF2 glycoprotein of EBV, wherein the antibody binds to epitopes within amino acids 41 to 160 of SEQ ID NO: 25 and is capable of reducing the infectivity of EBV to human epithelial cells.

[0200] (16) The antibody described in item 15, wherein the antibody binds to epitopes in amino acids 80 to 160 or 129 to 143 of SEQ ID NO: 25 and is capable of reducing the infectivity of EBV to human epithelial cells.

[0201] (17) The antibody described in item 15 or 16, wherein the antibody is

[0202] (a) An antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 8;

[0203] (b) An antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 16.

[0204] (c) An antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 24; or

[0205] (d) An antibody that binds to the same epitope as any of the antibodies in (a) to (c) of the EBV BILF2 protein shown in SEQ ID NO: 25.

[0206] (18) The antibody described in any one of items 15 to 17, wherein the antibody is

[0207] (a) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 3, and

[0208] The light chain variable region comprises a light chain CDR1 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 6.

[0209] (b) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 9, a heavy chain CDR2 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 10, and a heavy chain CDR3 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 11, and

[0210] The light chain variable region comprises a light chain CDR1 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 12, a light chain CDR2 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 13, and a light chain CDR3 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 14;

[0211] (c) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 17, a heavy chain CDR2 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 18, and a heavy chain CDR3 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 19, and

[0212] A light chain variable region comprising a light chain CDR1 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 20, a light chain CDR2 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 21, and a light chain CDR3 having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 22; or

[0213] (d) An antibody that binds to the same epitope as any of the antibodies in (a) to (c) of the EBV BILF2 protein shown in SEQ ID NO: 25.

[0214] (19) The antibody described in any one of items 15 to 18, wherein the antibody is

[0215] (a) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and

[0216] The light chain variable region comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0217] (b) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11, and

[0218] The light chain variable region comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 14.

[0219] (c) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19, and

[0220] A light chain variable region comprising light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 20, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 21, and light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 22; or

[0221] (d) An antibody that binds to the same epitope as any of the antibodies in (a) to (c) of the EBV BILF2 protein shown in SEQ ID NO: 25.

[0222] (20) The antibody of any one of items 15 to 19, wherein the epitope is determined by PepSpot analysis and / or deletion analysis of the extracellular domains covering amino acids 18 to 212 of BILF2 as shown in SEQ ID NO:25.

[0223] (21) The antibody of any one of items 15 to 20, used in a method of passive immunization against EBV.

[0224] (22) The antibody of any one of items 15 to 21, used in a method for reducing the infectivity of EBV to human epithelial cells.

[0225] (23) The antibody of any one of items 15 to 22, used in a method for preventing EBV infection of human epithelial cells.

[0226] (24) A method for diagnosing whether a human subject has antibodies that reduce the infectivity of EBV to human epithelial cells, the method comprising determining whether antibodies in a serum sample obtained from the subject competitively bind to any of the antibodies in items 15 to 20.

[0227] ***

[0228] Those skilled in the art will recognize, or can determine, many equivalent embodiments of the invention described herein using only conventional experiments. This invention is intended to cover these equivalent embodiments.

[0229] Unless otherwise stated, the following terms, including specifications and items, as used herein have the definitions given below.

[0230] It must be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein include plural references. Thus, for example, reference to “a reagent” includes one or more such different reagents, and reference to “the method” includes equivalent steps and methods known to those skilled in the art, which may be modified or substituted for the methods described herein.

[0231] Unless otherwise stated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalent embodiments of the specific embodiments of the invention described herein using only conventional experiments. This invention is intended to cover these equivalent embodiments.

[0232] The term “and / or” as used herein includes “and,” “or,” and “all or any other combination of the elements connected by the term.”

[0233] As used herein, the term “about” or “approximately” means within 20%, preferably within 10%, and more preferably within 5%, of a given value or range. However, it also includes specific numbers, such as about 20 including 20.

[0234] Throughout this invention and in its following documents, unless the context otherwise requires, the word “comprise” and its variations such as “comprises” and “comprising” will be understood to mean including the integers or steps stated herein, or a set of integers or steps, but not excluding any other integers or steps, or a set of integers or steps. When the term “comprising” is used herein, it may be replaced by the terms “containing” or “including,” or sometimes by the term “having.”

[0235] When this document uses "composed of...", it means that any element, step, or component not specified in the project elements is excluded. When this document uses "essentially composed of...", it does not exclude materials or steps that do not substantially affect the essential characteristics and novelty of the project.

[0236] In each instance of this document, any one of the terms “including,” “essentially composed of,” and “composed of” may be replaced by any one of the other two terms.

[0237] It should be understood that this invention is not limited to the specific methodologies, schemes, materials, reagents, and substances described herein, as these can be modified. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the terms used.

[0238] Other embodiments are described in the following items. Furthermore, where features or aspects of the invention are described in the form of the Markush group, those skilled in the art will recognize that the invention is therefore also described in the form of any single member or subgroup of the Markush group.

[0239] Of course, whenever a composition or antibody is disclosed herein for use in a treatment method or similar method, it means that such a composition or antibody can also be used in a treatment method. For example, such disclosure also applies to treatment methods that include the administration of such a composition or antibody.

[0240] All publications referenced in this specification (including all patents, patent applications, scientific publications, manufacturer's instructions, guides, etc.) are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to any prior art based on such disclosure. To the extent that any material incorporated by reference contradicts or is inconsistent with this specification, this specification supersedes any such material.

[0241] V. Attached Figure Description

[0242] Figure 1 The BILF2 monoclonal antibody and control antibody, tested for neutralization properties using EB-VLP and the human B cell line Daudi cells, are shown. Note that RLU = relative optical units; normalized to 100%.

[0243] Figure 2 The BILF2 monoclonal antibody and control antibody are shown in the test of neutralizing properties on HEK293 cells (a substitute for human epithelial cells) using 2089 EBV stock solution. Note that % of infected HEK293 cells are normalized to 100%.

[0244] Figure 3 The image shows human serum from EBV-positive individuals with serum antibodies that compete with neutralizing monoclonal antibodies and interfere with their binding to discrete epitopes on the BILF2 glycoprotein.

[0245] Figure 4Serum antibodies against BILF2, detected in sandwich ELISA, were shown after immunizing BALB / c mice with four different types of BILF2 antigen (BILF2 protein, BILF2 EV) or nucleic acid encoding BILF2 (BILF2 DNA, BILF2 mRNA).

[0246] Figure 5 The titers of EBV neutralizing antibodies in mouse serum immunized with four different types of BILF2 antigen (BILF2 protein, BILF2 EV) or nucleic acid encoding BILF2 (BILF2 DNA, BILF2 mRNA) are shown in EBV stock solution and HEK293 target cells.

[0247] Figure 6 The correlation between individual serum samples from 28 mice immunized three times with four different types of BILF2 antigen and the two assays is shown. A graph was plotted showing the relationship between the titer measured in the BILF2-specific sandwich ELISA (x-axis) and the titer obtained in the VNT assay (y-axis). Statistical analysis showed a Spearman r value of 0.7949, a 95% confidence interval of 0.5921–0.9030, and a two-tailed p-value < 0.0001, indicating a strong correlation between the data obtained in the two assays.

[0248] VI. Sequence

[0249] This invention provides and refers to the following sequences:

[0250]

[0251]

[0252]

[0253]

[0254]

[0255] .

[0256] VII. Examples

[0257] Example 1: Generation of EB-VLP

[0258] The inventors of this invention have engineered a vaccine candidate called Epstein-Barr virus-like particle (EB-VLP). EB-VLP is an enveloped vesicle mimicking the composition of EBV particles and contains many different viral proteins, such as glycoproteins exposed on the vesicle membrane, various envelope proteins, and numerous components forming the viral capsid contained within the lumen of the EB-VLP. EB-VLP is released in a stable form from production cells carrying viral genetic information. To induce the synthesis of EB-VLP in a production cell line and its release into the cell culture supernatant, a viral lysis phase in the cells is induced by adding a drug to the production cells, which are engineered to enter the lysis phase of EBV and release EB-VLP. EB-VLP is harvested from the cell culture supernatant and purified, subsequently used as the active pharmaceutical ingredient in an EBV vaccine candidate (see WO2012 / 025603).

[0259] Example 2: Protein composition analysis of EB-VLP

[0260] The inventors of this invention analyzed the protein composition of highly purified EB-VLP using mass spectrometry. As expected, EB-VLP was found to contain more than 50 viral proteins from all three classes of viral particle proteins (enveloping proteins, i.e., glycoproteins, envelope, and capsid).

[0261] Example 3: Analysis of the immune response to EB-VLP

[0262] The aim of immunizing mice and rats with EB-VLP was to obtain monoclonal antibodies that recognize many different viral proteins and to identify antibodies with neutralizing properties against viral (glycoproteins) present on the surface of EB-VLP.

[0263] Specifically, mice and rats were immunized with EB-VLP in a primiparous-booster regimen, the EB-VLP being purified according to standard methods. In short, EB-VLP was harvested from the cell culture supernatant of production cells carrying a genetically modified EBV genome, which lacks several EBV genes, one of which is essential for packaging genomic viral DNA into infectious viral particles. In these cells, EB-VLP production can be induced by a conditional molecular switch that, upon activation, induces a so-called EBV lysis phase, thus turning the production cells into EB-VLP factories. The production cell lines released EB-VLP into the supernatant for up to four days. When collecting the supernatant, cells and cell debris were removed by low-speed centrifugation, and EB-VLP was precipitated by ultracentrifugation. EB-VLP was further purified by flotation in a discontinuous iodixanol (Optiprep, Sigma-Aldrich) density gradient and ultracentrifugation in an equilibrium density gradient. EB-VLP was collected by fractionation as described in (Roessler et al., 2023). Animals received 10 via subcutaneous and intraperitoneal injections 9 Up to 10 10 One EB-VLP particle, with CpG as an adjuvant, is administered using a primary-boost regimen at two to three-month intervals.

[0264] The sera from each rodent were also used to detect protein spot blots, which covered a complete library of viral proteins purified from HEK293 cells transiently transduced with expression plasmids from 86 individuals (plus appropriate controls).

[0265] In addition, the rodent serum was used to detect a single EBV glycoprotein on HEK293 or HEK293T cells by quantitative flow cytometry, the cells expressing a single, isolated EBV glycoprotein on their cell surface.

[0266] In addition to these proteome and glycoprotein screenings for other antigens, the inventors discovered that two viral glycoproteins, gp350 / 220 and BILF2, are two major targets of the humoral immune response in mice.

[0267] gp350 / 220 are transmembrane proteins embedded in the membrane of infectious EBV particles, and are also present in the EB-VLP membrane (using mass spectrometry and nanofluid analysis).

[0268] In contrast, BILF2 is also one of the 13 known or predicted glycoproteins of EBV, first described in 1990 (Mackett et al., 1990), but has not attracted much interest in the EBV field.

[0269] Therefore, very little is known about BILF2, and no function has been determined. Occasionally, sporadic information about BILF2 appears in the prior art, suggesting that BILF2 may, in principle, be a target of the human immune response. Notably, the BILF2 protein sequence is 100% conserved in all prototypical EBV strains (such as B95.8, AG876, and M81), indicating that this glycoprotein plays an important role in the biology of EBV and may play a significant role in pathogenesis and immunity.

[0270] The inventors immunized several BALB / c mice and Wistar rats with EB-VLP, primarily to generate monoclonal antibodies against the antigens contained in the active pharmaceutical ingredient of an EBV vaccine candidate. In preliminary screening following somatic cell fusion of mouse spleen cells, the inventors observed a very high frequency of hybridoma supernatants containing antibodies against EBV (see Table 1). The inventors found that approximately 58% of these hybridoma supernatants contained antibodies against gp350 / 220, while 24% contained BILF2-specific antibodies. The remaining supernatants contained antibodies against other EBV glycoproteins or viral proteins that were not immediately recognizable.

[0271] .

[0272] Example 4: Analysis of immune response to extracellular vesicles (EVs) expressing only BILF2.

[0273] Similarly, the inventors immunized BALB / c mice with extracellular vesicles (EVs) expressing only the BILF2 protein (embedded in its membrane) (referred to as BILF2-EVs). To generate BILF2-EVs, HEK293 cells were transduced with a codon-optimized BILF2 expression vector to express BILF2 ectopically. As expected, the manipulated cells released large quantities of EVs with the BILF2 protein on their surface into the cell culture supernatant. Similar to EB-VLP, BILF2-EVs were harvested, purified, and concentrated for use as an antigen in immunization experiments with the aim of generating BILF2-specific monoclonal antibodies.

[0274] Animals received 10 via subcutaneous and intraperitoneal injections 9 Up to 10 10 One BILF2-EV, with CpG as an adjuvant, was administered using a prim-boost regimen with intervals of two to three months.

[0275] Example 5: Sequencing and characterization of antibodies against BILF2

[0276] In the two main methods of immunization using EB-VLP or BILF2-EV, the inventors cloned rat and mouse monoclonal antibodies with BILF2 specificity and different IgG isotypes.

[0277] Specifically, according to conventional hybridoma techniques, spleen cells from immunized animals were used for somatic cell fusion. Supernatants from hybridoma cell cultures were used to screen for antibodies specific to EBV glycoproteins. For this purpose, the inventors used flow cytometry and a mixture of cells from an induced EB-VLP production cell line and an equal volume of control HEK293 cells. To distinguish the two cell populations, one was stained with celltraceviolet (CTV, ThermoFisher Scientific) before incubating the cell mixture with the hybridoma supernatant, and then incubated with a secondary antibody conjugated to an appropriate fluorescent dye after washing. A suitable gating strategy enabled the identification of hybridomas specific to viral glycoproteins.

[0278] To identify antigen specificity, HEK293 cells expressing a single viral glycoprotein or a combination of two viral glycoproteins on their surface were generated by transient or stable transfection with expression vectors encoding individual EBV glycoproteins or combinations thereof. These cells were used in a manner similar to that described in the primary screening above. For candidates of interest, multiple rounds of limiting dilution subcloning of hybridoma cells were performed to achieve monoclonalness and to identify antibody specificity for the selected candidates. In the primary screening, most hybridomas released antibodies against gp350 / 220 or BILF2; among numerous other specificities, some subclones were selected for monoclonalness.

[0279] sequencing

[0280] Specifically, total RNA was isolated from selected monoclonal hybridoma cell lines using standard protocols. cDNA was generated using a 5' RACE system (ThermoFisher Scientific) for rapid cDNA end amplification, followed by PCR amplification using isotype-specific primer pairs. The PCR products were sequenced (Sanger sequencing, MWG Eurofins), and the resulting sequences were analyzed and annotated using algorithms provided by Abysis (http: / / www.abysis.org / abysis, UCL).

[0281] The DNA sequences of the RT-PCR amplification products were analyzed using Abysis software to identify and annotate the predicted CDRs according to Kabat (http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0282] KD value determination of anti-BILF2 antibody

[0283] HEK293 cells expressing BILF2 at moderately stable levels were transiently transduced using an expression plasmid encoding BILF2. These cells were mixed with an equal volume of control HEK293 cells, and the control cells were stained with celltrace violet (CTV, ThermoFisher Scientific) before incubation with serial dilutions of the purified monoclonal antibody against BILF2 (starting concentration 40 μg / mL) listed in Table 2. After incubation at 37°C for 60 min, the cell mixture was washed and incubated with a secondary antibody conjugated to an appropriate fluorescent dye and containing anti-rat or mouse IgG. Flow cytometry was performed using a Fortessa instrument (Becton Dickinson), and HEK293 control cells and BILF2-expressing cells were gated according to CTV staining to obtain the mean fluorescence intensity (MFI) for both cell types. The MFI value obtained from the BILF2-expressing cells was subtracted from the MFI value of the control HEK293 cells. After baseline correction, the results for all diluted samples were plotted and analyzed using a nonlinear fitting (S-shaped, 4PL) model provided by Prism (version 10.2, Graphpad). The average KD was obtained from three to four independent experiments based on the initial antibody concentration. 50 The values ​​are shown in Table 2.

[0284] Epitope mapping of BILF2 protein-specific monoclonal antibodies

[0285] Two different methods were used.

[0286] (i) A peptide library covering the BILF2 amino acid sequence, along with appropriate controls, was spotted and arrayed on a nitrocellulose membrane and incubated with the monoclonal antibodies shown in Table 2. The peptide library consisted of 60 peptides, each with 15 amino acids. Binding to each peptide was detected by infrared fluorescence using secondary antibodies (fluorescently labeled antibodies), which was recorded using a CCD camera. The four monoclonal antibodies produced clear signals.

[0287] (ii) Transiently express full-length BILF2 protein or a BILF2 mutant in HEK293 cells, wherein the mutant has a 40-amino acid deletion in each of the extracellular domains of BILF2 (covering amino acids 18 to 212 of BILF2 as shown in SEQ ID NO: 25). Incubate cells with an appropriately diluted monoclonal antibody and monitor the binding of the antibody to the surface of BILF2-expressing cells by flow cytometry using a secondary antibody conjugated with an appropriate fluorescent dye against rat or mouse IgG.

[0288]

[0289] It is noteworthy that in the antibody competition analysis, antibodies 27F5 and 28H3 partially competed for epitope binding, which may indicate that their epitopes may partially overlap, while antibody 9B8 did not compete for epitope binding with either antibody 27F5 or 28H3.

[0290] Therefore, when the antibodies of the present invention bind to the epitopes within amino acids 40-160 of BILF2 as shown in SEQ ID NO: 25, these antibodies can neutralize EBV infection of epithelial cells. Preferably, the epitopes are located within amino acids 80-160 or 129-143 of BILF2 as shown in SEQ ID NO: 25.

[0291] Example 6: Determination of the effect of anti-BILF2 antibody on reducing or blocking EBV infection in B cells or epithelial cells

[0292] Two different VNTs were used to evaluate the neutralizing properties of the monoclonal antibodies. To test them against EBV target cells, B cells and epithelial cells, namely Daudi and HEK293 cells, were used respectively.

[0293] Daudi cells were used as human B cells in combination with EB-VLP, as described by Roessler et al. (Roessler et al., 2023). For this purpose, Daudi cells were stably transduced using a retroviral vector encoding a larger fraction of the splitting NanoLuc luciferase (referred to as LgBiT), which forms a C-terminal fusion protein with human CD63. Similarly, EB-VLP was assembled with a smaller fraction of the splitting NanoLuc luciferase, referred to as HiBiT. (Roessler et al., 2023) describes the materials and setup for this analysis in detail. Briefly, 1 to 2 μg of purified monoclonal antibody or 40 μL of raw hybridoma cell culture supernatant in 40 μL PBS was incubated with 30 μL of EB-VLP at 37°C for 60 min. 4 × 10⁴ μg of EB-VLP was added to a 50 μL volume. 4 Daudi cells were incubated at 37°C for 4 hours to allow for cell adhesion, fusion, and uptake of EB-VLP. NanoLuc luciferase activity in Daudi cells incubated with EB-VLP was quantified as described (Roessler et al., 2023).

[0294] HEK293 target cells were used as epithelial cell targets for EBV. 1–2 μg of purified monoclonal antibody in 40 μL of raw hybridoma cell culture supernatant or 40 μL of PBS was incubated with 50 μL of EBV stock solution for 60 minutes. Each mL of the 2089 EBV stock solution encoding GFP contained 1 × 10⁻⁶ oz. 6Green Raji units (GRU) (Steinbrück et al., 2015). The mixture was added to 500 μL of RPMI 1640 containing 10% fetal bovine serum and common additives at a concentration of 2 × 10⁻⁶. 4 HEK293 cells were mixed and seeded in individual wells of 48-well cluster plates for 72 hours. Cells were trypsinized, resuspended, and analyzed by flow cytometry using a Fortessa instrument (Beckton-Dickinson) and a high-throughput sample unit. Gated GFP-positive cells were used, and the mean percentage (approximately 15%) of GFP-positive cells in controls without hybridoma cell culture supernatant or purified monoclonal antibody was set as 100% for normalization.

[0295] B-cell infection

[0296] The inventors tested whether the monoclonal antibody interfered with or neutralized viral infection in human B cells, which are the primary targets of EBV in vivo and in vitro.

[0297] In virus neutralization assays using EB-VLP as the viral stock solution and Daudi cells as the target of susceptible human B cells, the purified antibody (or the hybridoma supernatant before purification) did not reduce or block infection. Figure 1 The generation and purification of EB-VLP, as well as a validated Daudi cell-based EBV neutralization assay, are described in detail in (Roessler et al., 2023).

[0298] Epithelial cell infection

[0299] EBV also infects some human epithelial cells in vitro and is consistently associated with two EBV-positive tumor solids in patients with epithelial cancer. All nasopharyngeal carcinoma (NPC) cases and approximately 10% of gastric cancer (GC) cases are EBV-positive, demonstrating that EBV can infect human epithelial cells in vivo. EBV can also be found in the epithelial cells of the tongue in immunocompromised individuals (such as patients with advanced AIDS), where EBV is associated with a syndrome known as oral hairy leukoplakia, characterized by EBV replication in the squamous epithelial cells of this organ. In EBV-positive individuals with viral control, EBV is not detected in normal epithelial cells of the nasopharynx or tongue, suggesting that EBV-controlled individuals develop an immune response to prevent or eliminate EBV infection of epithelial cells.

[0300] Several epithelial cell models for EBV infection exist, such as AGS cells and the HEK293 cell line, which have been widely used as models for epithelial cell entry of infectious EBV or for cell-based fusion assays to study the interaction between viral ligands and cell receptors. The inventors used a viral stock solution of the EBV 2089 strain (Delecluse et al., 1998), which has a recombinant viral genome encoding the GFP gene, to facilitate quantitative monitoring of cell infection. For the epithelial cells, the inventors used HEK293 cells as previously described (Janz et al., 2000).

[0301] The inventors infected HEK293 cells with a calibrated EBV 2089 strain viral stock solution, resulting in approximately 12 to 17% of the target cells expressing GFP after three days. The inventors found that this infection rate was within the range reflecting the linear dose-response in this model. When the viral stock solution was used to infect Raji cells, the viral dose corresponded to approximately 5 × 10⁻⁶ as defined by Steinbrück et al. 4 One green Raji unit (GRU) (Steinbrück et al., 2015). In virus neutralization assays using a single monoclonal antibody against BILF2 and a control antibody, we used this dose contained in 50 μL of EBV stock solution. The virus stock solution was incubated with varying amounts of antibody diluted in PBS at a total volume of 90 μL at 37°C for 60 to 90 minutes. The virus-antibody mixture was then transferred to 2 × 10⁶ cells / mL of cell culture medium in 500 μL. 4 In HEK293 cells, the cell culture medium was placed in the wells of a 48-well cluster plate and incubated for three days. The percentage of GFP-positive cells was then analyzed by flow cytometry.

[0302] In this infection model, the inventors found that an established BILF2-specific monoclonal antibody, 28H3, neutralized EBV to a degree comparable to that of two neutralizing antibodies against the viral gH / gL glycoprotein complex. Figure 2 The 25G11 and 26B5 antibodies are comparable. Two other BILF2 antibodies interfere with epithelial cell infection to some extent. Figure 2 Among them, 27F5 and 9B8 were found, while the remaining BILF2-specific antibodies did not show a phenotype.

[0303] The gH / gL complex is known to be essential for EBV infection of B cells and epithelial cells, suggesting that antibodies against BILF2 and gH / gL may have an adductal effect in eliminating human epithelial cell infection.

[0304] control antibody

[0305] The inventors also tested control antibodies in this infection model. Three monoclonal antibodies against gp42 of EBV neutralized B-cell infection (…). Figure 1 However, it has no significant function in preventing epithelial cell infection. Figure 2 This is because gp42 physically contacts HLA class II molecules that are present only on B cells and not on epithelial cells. Therefore, gp42 is nonfunctional in the pre-fusion complex targeting different receptors (not HLA molecules) on epithelial cells. Consequently, gp42-specific antibodies are not expected to block infection of epithelial cells (Chesnokova et al., 2009).

[0306] Another control antibody (1D2) binds to the cell surface marker CD47, which is expressed very strongly on HEK293 cells. 1D2 also does not interfere with EBV infection in epithelial cells. Figure 2 This indicates that the protein corona of antibodies covering the surface of HEK293 cells does not prevent EBV from infecting these cells.

[0307] Based on these observations, the viral BILF2 glycoprotein appears to have a unique function in the infection process of epithelial cells. Antibodies against BILF2 can neutralize EBV infection in epithelial cells rather than B cells, highlighting the specific role of antibodies in combating EBV.

[0308] Example 7: Sequencing of BILF2 antibody

[0309] The light and heavy chain variable regions of three neutralizing monoclonal antibodies, namely monoclonal antibodies 27F5, 28H3 and 9B8, were determined; see Table 3.

[0310] .

[0311] Example 8: Testing for BILF2 antibodies in the serum of EBV carriers

[0312] The inventors became interested in another related finding from an analysis of human serum. Using Western blot analysis of a complete proteomic library covering 86 EBV proteins, the inventors tested the serum of approximately 100 healthy human volunteers. They found that serum antibodies against BILF2 were the most prevalent in the donor group's serum, and were more common than those against gp350 / 220 (Table 4). This information was obtained from the serum of EBV-positive adults who were apparently under control for viral infection.

[0313] The inventors also discovered that other viral glycoproteins, such as gp42 (BZLF2), gH, gL, and gB (BALF4), were detected in only a small percentage of human serum samples.

[0314] This is a surprising finding because their relative protein abundance in EB-VLP is not correlated with the prevalence of EBV glycoprotein-specific human serum antibodies. Therefore, it appears that BILF2 is the least abundant viral glycoprotein found in EB-VLP as listed in Table 4, but BILF2 ranks first in terms of immunogenicity in humans, even surpassing gp350 / 220, which is the most abundant viral glycoprotein in that ranking.

[0315] As already noted, in mice and rats immunized with EB-VLP, the inventors found that antibodies against gp350 and BILF2 were dominant in the humoral immune response of the animals, reflecting the situation in human EBV controllers.

[0316]

[0317] The ranking order #1 indicates the most abundant EBV protein among the 53 EBV proteins detected by the iBAQ algorithm;

[0318] Compared to gp350 / 220, which served as a reference, the abundance of BILF2 decreased by 84 times;

[0319] Relative fluorescence units (maximum = 2.0; after background correction);

[0320] EBV: Found only in EBV strains or related non-human primate EBV;

[0321] Common: It has homologs in other herpesviruses.

[0322] Example 9: Competitive analysis between the BILF2 antibody of the present invention and human serum antibody

[0323] The inventors also investigated whether the BILF2-specific monoclonal antibody of the present invention binds to epitopes that also produce antibodies against EBV-positive individuals.

[0324] If human serum contains antibodies that bind to an epitope that is also recognized by three neutralizing monoclonal antibodies, 27F5, 28H3, or 9B8, and a non-neutralizing antibody, then competitive analysis can help identify such human antibodies that may be important for preventing infection of human epithelial cells.

[0325] The inventors designed a competition assay using a population of HEK293 cells, comprising approximately 25 to 30% BILF2-positive and BILF2-negative cells. For this purpose, HEK293 cells were transiently transfected with an expression plasmid encoding BILF2. Only a subset of the cells (approximately 30%) expressed the BILF2 protein on their surface, while the majority of the cells did not.

[0326] cells and Figure 3 The serially diluted serum, as shown, was incubated at room temperature for 60 minutes from three EBV-positive individuals (top row inset) and three EBV-negative individuals (bottom row inset) to allow human BILF2-specific serum antibodies to bind to all epitopes on the EBV glycoprotein.

[0327] Wash cells with 1% BSA, then with... Figure 3 The four individual monoclonal antibodies labeled with Alexa 488, as shown, were incubated together. The concentrations of the four fluorescently labeled antibodies were carefully adjusted to provide a robust signal without competing with the human antibody that binds to the BILF2 epitope.

[0328] After incubation at 4°C for 20 minutes, cells were washed and analyzed by flow cytometry using a BD Fortessa instrument. Serial dilutions of the six sera were plotted on the x-axis, and the percentage of Alexa 488 positive cells was shown on the y-axis.

[0329] High concentrations of serum from EBV-positive individuals prevent the binding of BILF2-specific monoclonal antibodies, indicating that the epitopes of these antibodies are blocked by human serum antibodies, making it impossible for BILF2-specific monoclonal antibodies to access them.

[0330] Analysis of the serum of three EBV-positive individuals showed decreased binding of all four monoclonal antibodies to BILF2-positive cells, varying with human serum antibody concentration. With successive serum dilutions, the BILF2 epitope became increasingly accessible, leading to an increase in Alexa488-positive cells, as monoclonal antibodies can bind to their corresponding epitopes in the absence of human antibodies.

[0331] exist Figure 3 In the study, for serum from EBV-positive individuals, all four monoclonal antibodies showed reduced binding at low serum dilutions (1:8, 1:16, etc.), while no reduction was observed at high serum dilutions (1:256).

[0332] This effect was not observed in sera from three EBV-negative individuals, indicating that the competitive effect at low serum dilutions (i.e., high antibody concentrations) is specific and demonstrates the binding of human antibodies to the BILF2 epitope. Therefore, the sera from EBV-negative individuals did not contain antibodies that competitively bind to the BILF2-positive cell portion with the BILF2-specific monoclonal antibody, as shown by the almost flat horizontal line in the inset below.

[0333] Three of the four monoclonal antibodies used in the competitive analysis, namely 27F5, 28H3, and 9B8, have neutralizing properties. Figure 2 This indicates that human serum antibodies can also neutralize EBV to prevent and interfere with its infection of human epithelial cells.

[0334] As demonstrated in virus neutralization tests, antibody 26G5 does not interfere with EBV infection of epithelial cells.

[0335] Example 10: Immunizing BALB / c mice with four different BILF2 antigens

[0336] Four different types of BILF2 antigens were prepared to immunize four groups of mice using a primary-booster-booster regimen. Two BILF2 antigens encode nucleic acids and two different types of BILF2 proteins comprise (i) expression vector plasmid DNA; (ii) a preparation of synthetic mRNA; (iii) purified, adjuvanted BILF2 protein; and (iv) a preparation of extracellular vesicles (EVs) carrying BILF2 protein (in addition to cell-derived proteins) on their surface.

[0337] Regarding (i)

[0338] A codon-optimized synthetic DNA encoding BILF2 was designed to express the viral protein BILF2 in its native form. The synthetic DNA fragment was cloned into a conventional vector plasmid, and its sequence was confirmed. The coding sequence for BILF2 was amplified using two suitable PCR primers and inserted into a version of the expression vector plasmid pVAX1. After transformation of *E. coli* strain DH5alpha and selection with kanamycin, the final pVAX1-based BILF2 expression plasmid, named 7617.SA1, was constructed and sequenced to confirm its identity. Plasmid DNA was isolated from a large number of cultures of *E. coli* clones using Qiagen's Endofree Plasmid Maxi kit. The endotoxin concentration was determined to be 0.03 EU / 100 μL at a plasmid DNA concentration of 1 μg / μL. 100 μg of plasmid DNA was administered intramuscularly to each mouse as a single dose. The sequence of the portion encoding BILF2 in plasmid 7617.SA1 is provided in SEQ ID NO: 30.

[0339] Regarding (ii)

[0340] A custom-designed mRNA was ordered from PackGene Biotech Inc., which encodes a codon-optimized BILF2 open reading frame according to specifications according to the present invention. The mRNA was produced using T7 RNA polymerase and N1-methylpseudouridine (…). A combination of triphosphates and three unmodified ribonucleotide triphosphates, transcribed from appropriately designed plasmid DNA as a template (after appropriate restriction enzyme cleavage), is used for RNA synthesis to bypass the innate immune response and improve in vivo translation efficiency. A Cap-1 structure is added to the 5' end, and the RNA is encapsulated in lipid nanoparticles (LNPs) at a concentration of 0.2 mg RNA / mL. The formulation contains <1 EU / mL endotoxin and is frozen in aliquots and stored at -80°C for later use. The formulation is diluted, and mice are immunized with 10 μg of RNA contained in 100 μL of the solution via intramuscular administration. The sequence of the synthetic mRNA encoding BILF2 is provided in SEQ ID NO: 31.

[0341] Regarding (iii)

[0342] BILF2 protein was prepared from the cell culture supernatant of HEK293T cells transiently transfected with an expression plasmid encoding modified BILF2. The expression plasmid 7703.TA1 encodes a codon-optimized open reading frame of BILF2 with a C-terminal TEV cleavage site, followed by an EBNA1 epitope and a tag consisting of six histidine residues. The single transmembrane domain (ΔTM) of BILF2 was deleted to facilitate the release and secretion of the modified BILF2 protein into the supernatant of transfected cells. The backbone of expression plasmid 7703.TA1 is a pVAX1 vector modified to carry an SV40 promoter / enhancer element, which also contains an SV40 DNA replication origin to allow plasmid DNA amplification and enhance BILF2 expression in transfected HEK293T cells. The sequence of the modified BILF2 coding portion of plasmid 7703.TA1 is provided in SEQ ID NO: 32.

[0343] HEK293T cells were transiently transfected with 7703.TA1 plasmid DNA, and the cell culture supernatant was harvested three days post-transfection. After low-speed centrifugation, Ni-beads (Pierce Inc.) were added in the presence of 10 mM imidazole. After overnight binding, the Ni-beads were thoroughly washed, and the bound protein was released by adding 0.5 M imidazole. The solution was concentrated using an Amicon Ultra 15 centrifuge filter (30 kDa cutoff), and the buffer was replaced to obtain a final concentration of 250 μg / mL BILF2 protein in PBS / 10% glycerol, as determined by a sandwich ELISA using highly purified BILF2 protein as a reference and standard. 60 nmol / mL ODN 1688 was added as an adjuvant. Mice were administered a single dose of 100 μL intramuscularly.

[0344] Regarding (iv)

[0345] Extracellular vesicles bearing the BILF2 protein on their surface were isolated from the cell culture supernatant of HEK293T cells three days after transient transfection with expression plasmid 7689.PA1. The 7689.PA1 DNA is sequence identical to that in 7617.SA1, encoding a codon-optimized BILF2 protein (SEQ ID NO: 30), but additionally, 7689.PA1 carries the SV40 DNA replication origin to enhance BILF2 protein expression in HEK293T cells after transient DNA transfection. EVs were concentrated by ultracentrifugation after two low-speed centrifugation steps to remove cells and cell debris, the precipitate was resuspended overnight in the remaining volume, and further purified by density-equilibrated centrifugation on a discontinuous iodixanol (Optiprep, Sigma-Aldrich) density gradient as described in detail (Roessler et al., 2023; Roessler et al., 2022). Fractions 2 and 3 were collected, diluted in PBS, and concentrated by sedimentation in an ultracentrifuge. After removing most of the supernatant, the EV was resuspended in the remaining volume. The BILF2 protein content was determined using a sandwich ELISA with highly purified BILF2 protein as a reference protein. The preparation was further diluted with PBS to obtain a final concentration of 22 μg / mL BILF2 protein, corresponding to approximately 5 × 10⁻⁶ μg / mL as determined by nanoparticle tracking analysis (NTA) using a ZetaView PMX110 instrument (Particle Metrix). 11 EV / mL. Before use, store BILF2 EV in aliquots at -80°C. Administer a single dose of 100 μL intramuscularly to mice.

[0346] Immunization regimen

[0347] On days 1, 31, and 61 of the study, approximately 7-week-old BALB / c mice received three intramuscular administrations of four antigens. Table 5 provides an overview of the antigens and immunization regimen. Administration followed a prime-booster-booster protocol. Blood was collected from all animals prior to immunization on day 1 of the study, then prior to the final immunization on day 61, and final blood samples were collected on day 75. Serum was prepared and stored at -20°C. Animal husbandry and care, immunization, and blood collection were performed by a commercial CRO (AurigonLabs Ltd, Pálya utca 2, 2120 Dunakeszi Hungary).

[0348] .

[0349] Detection and quantification of BILF2 specific serum antibodies

[0350] A BILF2-specific sandwich ELISA was established using BILF2 protein ectopically expressed and released into the cell supernatant in HEK293T cells. For this purpose, cells were transiently transfected with expression plasmid 7741.IA1, which encodes BILF2 with a carboxyl-terminal EBNA1 tag (EQGPADDPGEG) (SEQ ID NO: 33). After three days of culture in a medium containing only 2% fetal bovine serum, the cell culture supernatant was harvested, purified by low-speed centrifugation, and the concentration of BILF2 was analyzed. This analysis was performed in a separate ELISA, using a highly purified BILF2 protein formulation as a standard and reference, and BILF2 was detected using a BILF2-specific monoclonal antibody conjugated with HRP. The cell culture supernatant was found to contain 2.2 μg / mL BILF2 protein. For control purposes, an equivalent cell culture supernatant was produced from untransfected HEK293T cells. Both formulations were aliquoted and stored at -80°C until use.

[0351] To establish a BILF2-specific ELISA, each well of a 96-well cluster plate (F96 Maxisorp Nunc Immuno plate; Thermo Scientific #439454) was incubated overnight at 4°C with a capture antibody, namely an EBNA1-tagged specific monoclonal rat antibody, termed E1BS 1H4 (Grässer et al., 1994). After washing and blocking, 6 ng of EBNA1-labeled BILF2 protein, prepared as described above and diluted in 100 μL PBS, was added to each well to capture and immobilize the BILF2 protein. As a control and to assess the nonspecific binding of mouse serum immunoglobulins to the capture antibody or plastic surface in subsequent steps, supernatant from untransfected HEK293T cells was used accordingly. To allow protein binding, the 96-well cluster plate was incubated at room temperature for 4 hours. Afterward, the plate was washed to remove unbound protein.

[0352] Mouse serum was diluted in cell culture supernatant from untransfected HEK293T cells and incubated at room temperature for 60 minutes. Serial dilutions of the adsorbed serum were prepared in PBS in standard U-bottom 96-well cluster plates and transferred to plates containing immobilized BILF2 protein and a control. After overnight incubation at 4°C, the plates were washed, and mouse serum immunoglobulins were detected using HRP-conjugated anti-mouse antibodies. Binding was demonstrated using TMB (3,3',5,5'-tetramethylbenzidine), a substrate of HRP, and quantified by stopping the reaction after 10 minutes using a Clariostar microplate reader. OD measurements of the serial serum dilutions with and without BILF2 antigen were recorded, subtracted, and analyzed using Prism software (Graphpad Inc.) and its 4-parameter logic (4PL) model to characterize and calculate the midpoint of the sigmoid dose-response curve, with the corresponding serum dilution used as the antibody titer.

[0353] Detection and quantification of EBV neutralizing serum antibodies in the virus neutralization test (VNT)

[0354] The inventors learned that some monoclonal antibodies against BILF2 can inhibit EBV infection in HEK293 cells (Graham et al., 1977), but cannot inhibit EBV infection in Daudi cells (a human B cell line) (Klein et al., 1968).

[0355] Consistent with the “epithelial cell infection” described in Example 6 of this document, the concentration of neutralizing serum antibodies was determined by the VNT50 titer as measured in a virus neutralization assay using HEK293 cells. VNT50 refers to the dilution of a serum sample that blocked infection in 50% replicate inoculation. This measurement quantified the level of neutralizing antibodies that effectively antagonized EBV in the serum of BILF2-immunized mice. Serial dilutions of mouse serum were used as described herein, with an initial dilution of 1:8. The inventors determined the percentage of GFP-positive cells by flow cytometry and analyzed the data using Prism software (version 10.4, Graphpad Inc.) to calculate the VNT50 titer.

[0356] result

[0357] Anti-BILF2 serum antibodies were analyzed in mouse serology collected on day 1 before the first immunization, day 61 before the second immunization, and day 75 at the end of the experiment using ELISA. Titers are shown in... Figure 4 No BILF2-specific antibodies were detected in unimmunized mice, but significant antibody levels were observed in all animals except one (mice m31 immunized with BILF2 protein) following the primary-boost immunization on day 61. BILF2-specific antibody levels increased further on day 75, two weeks after the second booster immunization. Mice immunized with either BILF2 mRNA or BILF2 EV had significantly higher levels of BILF2 antibodies, averaging 1:6,112 or 1:4,219 on day 75, compared to averages of 1:704 or 1:809 for mice immunized with BILF2 DNA or BILF2 protein, respectively.

[0358] Using EBV stock solution and HEK293 cells as targets, neutralizing antibodies were analyzed in all serum samples collected at the end of day 75. The results, i.e., VNT50 titers, are shown in [data missing]. Figure 5 Titers higher than 1:10 were considered to contain neutralizing antibodies, consistent with titers in mouse serum collected on day 1 or other BALB / c control sera. At the end of the animal experiments, all mice in both groups immunized with either BILF2 mRNA or BILF2 EV possessed antibodies neutralizing EBV infection of HEK293 cells. Except for one mouse (m18), mice immunized with either BILF2 DNA or BILF2 protein did not produce detectable levels of EBV neutralizing antibodies.

[0359] Therefore, these results demonstrate that RNA encoding BILF2 and EVs displaying the BILF2 glycoprotein of EBV on their surface provide antibodies that neutralize EBV infection in HEK293 cells, which serve as a substitute for human epithelial cells. Accordingly, RNA encoding BILF2 and EVs displaying the BILF2 glycoprotein on their surface provide antibodies against the BILF2 glycoprotein, which reduce the infectivity of EBV to human epithelial cells, preferably preventing EBV infection of human epithelial cells.

[0360] Therefore, given these results, it is evident that attempts with existing techniques, such as using DNA expression plasmids targeting BILF2 (WO 2019 / 161163) or suggesting the use of the BILF2 protein as an immunogen (Mackett et al.), have failed to provide antibodies that neutralize EBV infection.

[0361] The inventors investigated whether the titers of the two different readout values ​​were correlated. Figure 6 The graphs for the two datasets are shown. Statistical analysis revealed a Spearman r value of 0.7949, a 95% confidence interval of 0.5921 to 0.9030, and a two-tailed p-value < 0.0001. This analysis indicates that high levels of BILF2 serum antibodies are strongly associated with neutralizing activity.

[0362] The results showed that in BALB / c mice, three administrations of 10 μg mRNA or unadjuvanted EV carrying 2.2 μg BILF2 protein via a primary-booster-booster regimen induced antibodies that inhibited EBV infection of HEK293 cells. Conversely, the concentration or mass of serum antibodies induced in mice immunized three times with 100 μg DNA encoding BILF2 or with 25 μg BILF2 protein adjuvanted with ODN 1688 failed to inhibit EBV infection of HEK293 cells.

[0363] It is noteworthy that lipid nanoparticles (LNPs) in mRNA-based vaccines act as adjuvants because LNPs enhance immunity through multiple mechanisms. LNPs promote B cell responses, induce key cytokines (particularly IL-6, which is crucial for follicular T helper cell differentiation), trigger innate immunity, and enhance antigen presentation and other stimulatory effects. In contrast, EVs carrying exogenous viral glycoproteins (here, BILF2 of EBV) inherently possess the potential to serve as highly immunogenic particulate antigenic structures. Unbound by theory, when combined with appropriate adjuvants, EVs are likely to elicit a stronger immune response against, for example, BILF2 compared to candidate vaccines using LNP-encapsulated mRNA.

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Claims

1. A composition comprising a) RNA encoding the BILF2 glycoprotein of EBV, or b) Extracellular vesicles (EVs) or EBV-like particles (VLPs) displaying the BILF2 glycoprotein of EBV on their surface. In a method of using the composition to induce an antibody against the BILF2 glycoprotein, the antibody reduces the infectivity of EBV to human epithelial cells.

2. The composition for use according to claim 1, wherein the antibody prevents EBV infection of human epithelial cells.

3. The composition of claim 1 or 2, wherein the EV or VLP expresses BILF2, and the BILF2 is expressed alone or in combination with one or more other EBV glycoproteins.

4. The composition of claim 3, wherein the other EBV glycoproteins may comprise gp350, gB, gp42, gH, gL, gM, gN, BMRF2, BDLF2, BDLF3, BILF1, and BARF1.

5. The composition of any one of the preceding claims, wherein the VLP further comprises one or more EBV structural proteins.

6. The composition of claim 5, wherein the one or more EBV structural proteins comprise a fusion protein (F), a matrix protein (M), or a nucleocapsid protein (N).

7. The composition of any one of the preceding claims further comprises one or more pharmaceutically acceptable carriers.

8. The composition of any one of the preceding claims further comprises one or more adjuvants.

9. The composition of any one of the preceding claims, used in a method of treating or preventing a disease.

10. The composition of claim 9, wherein the disease is cancer or an infection, the cancer is preferably an epithelial cancer, the infection is preferably EBV infection, and most preferably EBV infection of epithelial cells.

11. The composition of claim 9 or 10, wherein the disease is cancer, and the cancer is selected from: medulloblastoma, retinoblastoma, Burkitt lymphoma, Hodgkin lymphoma, oral cancer, skin cancer, basal cell carcinoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, gastric cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma, bladder cancer, anal cancer, chondrosarcoma, osteosarcoma, sarcoma, primitive neuroectodermal carcinoma (primitive neuroectodermal tumor (PNET)) and combinations thereof.

12. An antibody for a method of passive immunization against EBV, wherein the antibody targets the BILF2 glycoprotein of EBV, wherein the antibody binds to epitopes within amino acids 41 to 160 of SEQ ID NO: 25 and is capable of reducing the infectivity of EBV to human epithelial cells.

13. An antibody for use in a method of reducing infectivity of EBV for human epithelial cells, wherein the antibody is directed against the BILF2 glycoprotein of EBV, wherein the antibody binds to an epitope within amino acids 41 to 160 of SEQ ID NO: 25 and is capable of reducing infectivity of EBV for human epithelial cells.

14. An antibody for use in a method of preventing infection of EBV for human epithelial cells, wherein the antibody is directed against the BILF2 glycoprotein of EBV, wherein the antibody binds to an epitope within amino acids 41 to 160 of SEQ ID NO: 25 and is capable of reducing infectivity of EBV for human epithelial cells.

15. The antibody of any one of claims 12 to 14, wherein the antibody binds to an epitope within amino acids 80 to 160 or 129 to 143 of SEQ ID NO: 25 and is capable of reducing infectivity of EBV for human epithelial cells.

16. The antibody of any one of claims 12 to 15, wherein the antibody is (a) an antibody comprising a heavy chain variable region having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 8; (b) an antibody comprising a heavy chain variable region having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 16; (c) an antibody comprising a heavy chain variable region having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 23 and a light chain variable region having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 24; or (d) an antibody that binds to the same epitope in the EBV BILF2 protein set forth in SEQ ID NO: 25 as the epitope bound by the antibody of any one of (a) to (c).

17. The antibody of any one of claims 12 to 16, wherein the antibody is (a) an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain CDR3 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain CDR2 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain CDR3 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 11, and (b) an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain CDR2 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain CDR3 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 11, and a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 17, a heavy chain CDR2 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 18, and a heavy chain CDR3 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 19, and (c) an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 17, a heavy chain CDR2 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 18, and a heavy chain CDR3 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 17, a heavy chain CDR2 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 18, and a heavy chain CDR3 having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 19, and (d) an antibody that binds to the same epitope in an EBV BILF2 protein set forth in SEQ ID NO: 25 as the epitope bound by the antibody of any one of (a) to (c).

18. The antibody of any one of claims 12 to 17, wherein the antibody is (a) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and The light chain variable region comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

6. (b) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11, and The light chain variable region comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

14. (c) An antibody comprising a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19, and A light chain variable region comprising light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 20, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 21, and light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 22; or (d) An antibody that binds to the same epitope as any of the antibodies in (a) to (c) of the EBV BILF2 protein shown in SEQ ID NO:

25.

19. The antibody of any one of claims 12 to 18, wherein the epitope is determined by PepSpot analysis, amino acid substitution analysis and / or deletion analysis of the extracellular domains covering amino acids 18 to 212 of BILF2 as shown in SEQ ID NO: 25.

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