Patent Publication Number: US-2023159626-A1

Title: Epstein-barr virus antibodies and uses thereof

Description:
PRIORITY CLAIM 
     The present invention is a continuation of U.S. patent application Ser. No. 16/609,078, filed Oct. 28, 2019, which is a 371 national phase of International Patent Application No. PCT/US2018/030030, filed Apr. 27, 2018, which claims the benefit of U.S. Provisional Patent Application No. 62/491,945, filed Apr. 28, 2017, the content of which are incorporated herein by reference in their entirety. 
    
    
     STATEMENT OF GOVERNMENT INTEREST 
     The present invention was made with government support under Grant No. R21CA205106, awarded by U.S. Public Health Service. The Government has certain rights in the invention. 
    
    
     BACKGROUND 
     Epstein-Barr virus (EBV) infection is the causal agent of acute infectious mononucleosis (62, 63). Persistent EBV infection in immunodeficient individuals is associated with numerous epithelial and lymphoid malignancies, such as nasopharyngeal carcinoma, gastric carcinoma, Burkitt lymphoma, Hodgkin lymphoma, and post-transplant lymphoproliferative diseases (PTLD) (1). Transplantation is the treatment of choice for a variety of patients with end-stage organ failure or hematologic malignancies, or in need of reconstructive transplantation (1). Transplantation success depends entirely on potent immunosuppressive drugs to prevent stem cell/organ rejection. However, these drugs impose several serious side effects, including an increased risk of infection with or reactivation of Epstein-Barr virus (EBV), and the resultant development of PTLDs, which are aggressive, life-threatening complications (2, 3). Through the early 2000s, PTLD patients who had been EBV-naive prior to transplantation showed mortality rates of 50-90% for stem cell and solid organ transplants; while recent data suggest outcomes have improved, challenges remain. PTLDs usually develop in EBV-naive patients, particularly pediatric patients, who receive organs from EBV+ donors. A variety of non-standardized, non-specific treatments are used to treat EBV+ PTLD cases (4-9). Initial clinical management typically involves reduction of immunosuppression; however, this can lead to graft-versus-host disease. Other treatments including radiation/chemotherapy and excision of PTLD lesions all have undesirable side effects. Second-line treatment often includes antibodies (Abs) against the B cell antigen, CD20; however, this also targets healthy B cells, further weakening the immune system and exposing patients to other opportunistic infections. 
     In over 50 years of EBV vaccine research, few candidates have demonstrated partial clinical efficacy, and none have been efficacious enough to elicit sterilizing immunity and be licensed (24). Antibodies, whether elicited in the host naturally or via passive immunization, provide an effective first-line of defense against viral infection. 
     Thus, there is an urgent need for a novel EBV-specific therapy that targets EBV+ cells to neutralize EBV infection and prevent subsequent PTLD development in EBV-naive patients. 
     SUMMARY 
     In one aspect, this disclosure relates to an Epstein-Barr virus (EBV) antibody or an immunogenic fragment thereof. In some embodiments, the EBV antibody or an immunogenic fragment thereof specifically binds to EBV glycoprotein 350/220. In some embodiments, the EBV antibody or an immunogenic fragment thereof specifically binds to EBV glycoprotein 350 or one or more epitopes represented by SEQ ID NOs: 1-3. In some embodiments, the EBV antibody comprises a VH region comprising CDR-1, CDR-2, and CDR-3 represented by SEQ ID NOs: 4-19, 20-35, and 36-51, respectively. In some embodiments, the EBV antibody comprises a VL region comprising CDR-1, CDR-2, and CDR-3 represented by SEQ ID NOs: 52-67, 68-83, and 84-99, respectively. In some embodiments, the antibody is not antibody clone 72A1 or an antibody comprising the CDRs of antibody clone 72A1. In some embodiments, the EBV antibody is a monoclonal antibody. In some embodiments, the EBV antibody is a chimeric antibody, a human antibody, or a humanized antibody. 
     In another aspect, this disclosure relates to an immunogenic peptide comprising one or more gp350 epitopes having an amino acid sequence identical to or sharing at least 60% similarity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the immunogenic peptide comprises a first domain comprising an amino acid sequence having at least 60% similarity to SEQ ID NO:1, a second domain comprising an amino acid sequence having at least 60% similarity to SEQ ID NO: 2, and a third domain comprising an amino acid sequence having at least 60% similarity to SEQ ID NO: 3. In some embodiments, the immunogenic peptide comprises a first domain comprising an amino acid sequence having at least 60% similarity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and a second domain comprising a known immunogenic peptide such as keyhole limpet hemocyanin (KLH) peptide. 
     In another aspect, this disclosure relates to an EBV antibody-small molecule conjugate. The EBV antibodies disclosed herein can be conjugated to small molecules having activities against EBV-transformed cells. For example, the small molecules have anti-proliferative activities against EBV-transformed B lymphoma cells. In some embodiments, the small molecules are growth inhibitors of EBV infected B cells. In some embodiments, the small molecule is L 2 P 4 , 2-butynediamide, or a derivative thereof. In some embodiments, the small molecule is conjugated to the antibody via a linker or an adaptor. In some embodiments, the small molecule is conjugated to the constant region of the heavy chain or the light chain of the antibody. 
     In a related aspect, this disclosure relates to a pharmaceutical composition comprising the EBV antibody disclosed herein or an immunogenic fragment thereof, one or more epitopes represented by SEQ ID NOs: 1-3, one or more immunogenic peptides disclosed herein, or the EBV antibody-small molecule conjugate disclosed herein. The pharmaceutical composition can further comprise one or more pharmaceutically acceptable excipients. The pharmaceutical composition can be formulated into any suitable formulation depending on the administration route. 
     In another aspect, this disclosure relates to a method of neutralizing EBV infection. The method includes administering to a subject infected with EBV a therapeutically effective amount of the EBV antibody disclosed herein or an immunogenic fragment thereof, one or more epitopes represented by SEQ ID NOs: 1-3, one or more immunogenic peptides disclosed herein, the EBV antibody-small molecule conjugate, or the pharmaceutical composition described above. In some embodiments, the subject is human. 
     In another aspect, this disclosure relates to a method of preventing EBV infection. The method includes administering to a subject at an elevated risk of EBV infection a therapeutically effective amount of the EBV antibody disclosed herein or an immunogenic fragment thereof, one or more epitopes represented by SEQ ID NOs: 1-3, one or more immunogenic peptides disclosed herein, the EBV antibody-small molecule conjugate, or the pharmaceutical composition described above. In some embodiments, the subject is human. 
     In another aspect, this disclosure relates to a method of preventing a post-transplant lymphoproliferative disease (PTLD). PTLD is associated with EBV infection of B cells, either as a consequence of reactivation of the virus post transplantation or from primary EBV infection. The method includes administering to a subject who is a transplant recipient a therapeutically effective amount of the EBV antibody disclosed herein or an immunogenic fragment thereof, one or more epitopes represented by SEQ ID NOs: 1-3, one or more immunogenic peptides disclosed herein, the EBV antibody-small molecule conjugate, or the pharmaceutical composition described above. The administration can be before, during, and/or after the transplant. In some embodiments, the subject is a pediatric transplant recipient who is EBV naïve. In some embodiments, the subject is an adult transplant recipient. In some embodiments, the subject is human. 
     In another aspect, this disclosure relates to a method of treating an EBV-associated cancer. The method includes administering to a subject suffering from an EBV-associated cancer a therapeutically effective amount of the EBV antibody disclosed herein or an immunogenic fragment thereof, one or more epitopes represented by SEQ ID NOs: 1-3, one or more immunogenic peptides disclosed herein, the EBV antibody-small molecule conjugate, or the pharmaceutical composition described above. In some embodiments, the examples of EBV-associated cancer include but are not limited to Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, and nasopharyngeal carcinoma. In some embodiments, the subject is human. 
     In another aspect, this disclosure relates to a method of immunizing or vaccinating a subject against an EBV infection. The method includes administering to a subject suffering from an EBV infection a therapeutically effective amount of the EBV antibody disclosed herein or an immunogenic fragment thereof, one or more epitopes represented by SEQ ID NOs: 1-3, one or more immunogenic peptides disclosed herein, or the pharmaceutical composition thereof as described above. In some embodiments, the subject is human. 
     In another aspect, this disclosure relates to a method of inducing the production of neutralizing antibodies against a EBV in a subject. The method includes administering to a subject an effective amount of a gp350 epitope represented by SEQ ID NO: 3 or an immunogenic peptide comprising the gp350 epitope represented by SEQ ID NO: 3. In some embodiments, the subject is human. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS.  1 A- 1 D  show characterization of novel anti-gp350 mAbs.  FIG.  1 A : ELISA screening of hybridoma (HB) supernatants for anti-gp350-specific antibodies. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. nAb-72A1 at 10 μg/ml and 1× phosphate buffered saline (PBS) were used as positive and negative (not shown) controls, respectively. Bound antibodies were detected using HRP-conjugated anti-mouse IgG (1:2,000). Twenty-three HB clones with ELISA signals two times greater than those of PBS control were considered as positive hybridomas.  FIG.  1 B : Determining specificity of anti-gp350 producing hybridoma supernatant by immunoblotting with gp350-transfected stable CHO lysate. Western blot analysis was conducted on untransfected and pCAGGS-gp350 transfected CHO lysate. Anti-gp350 mAb 72A1 was used as a positive control (1:100) and anti-gp350 hybridoma clone supernatants were used at 1:50. Anti-mouse secondary antibody was used at 1:2000.  FIG.  1 C : Flow cytometric analysis of surface expression of gp350 protein on gp350 expressing CHO cells for all 23 HB clones. 10 6  CHO cells were transfected with 1 μg of pCAGGS-gp350. gp350 expressing cells were resuspended in PBS, stained with anti-gp350 mAb (1:250), which detects the gp350 ED, followed by secondary goat anti-mouse conjugated to AF488.  FIG.  1 D : Flow cytometric analysis of HB5, HB17 and HB19. 
         FIGS.  2 A and  2 B  show PROMALS3D multiple sequence alignment of VH ( FIG.  2 A ) and VL ( FIG.  2 B ) regions of 15 mAbs and nAb-72A1 (SEQ ID NOS:100-131). The highly variable complementarity determining regions (CDR) 1-3, indicated by black boxes, define the antigen binding specificity. The conserved framework regions (FR) 1-4 flank the CDRs. Consensus amino acid (aa) are in bold and upper case. Consensus predicted secondary structure (ss) symbols: alpha-helix: h and beta-strand: e. 
         FIGS.  3 A and  3 B  show neutralization activity of novel anti-gp350 mAbs against EBV-eGFP in Raji cells. EBV-eGFP was pre-incubated with 15 anti-gp350 mAbs at 10 μg/ml ( FIG.  3 A ) and 50 μg/ml ( FIG.  3 B ), followed by incubation with Raji cells for 48 h. EBV-eGFP+ cells were enumerated using flow cytometry. Anti-gp350 (nAb-72A1) and anti-gH/gL (E1D1) mAbs served as positive controls and non-neutralizing anti-gp350 (2L10) mAb served as negative control. 
         FIGS.  4 A- 4 C  show construction of chimeric gp350 nAbs.  FIG.  4 A : Construction of chimeric Ab.  FIG.  4 B : Cloning strategy of heavy chain and light chain variable regions into expression vectors.  FIG.  4 C : PCR amplification of heavy chain and light chain variable regions. 72A1 and clone 19 were used as examples of PCR amplification. 
         FIG.  5    illustrates an antibody-L2P4 conjugate. 
     
    
    
     DETAILED DESCRIPTION 
     Disclosed herein are EBV antibodies or immunogenic fragments thereof that specifically bind to gp350/gp220 or an epitope of gp350, immunogenic peptides comprising one or more gp350 epitopes, or one or more amino acid sequences having at least 60% similarity to one or more gp350 epitopes, EBV antibody-small molecule conjugates for treating or preventing EBV infection, in particular, in subjects receiving a transplant. In some embodiments, chimeric (human/mouse) anti-gp350 nAbs are conjugated to L2P4 to obtain an EBV-specific ADC that improves the therapeutic efficacy of treating EBV-associated PTLDs. L2P4 described by Jiang et al.,  Nature Biomedical Engineering  1: 0042 (2017), is an example of the small molecules encompassed by this disclosure. 
     The term “antibody” as used herein refers to an immunoglobulin molecule or an immunologically active portion thereof that specifically binds to, or is immunologically reactive with a particular antigen, for example, EBV gp350/gp220, or a particular domain or fragment of gp350/gp220. In some embodiments, the antibody disclosed herein specifically binds to one or more epitopes on EBV gp350. The epitopes can be neutralizing epitopes or immunodominant epitopes. In some embodiments, the epitope has an amino acid sequence selected from  253 TPIPGTGYAYSLRLTPRPVSRFL 275  (SEQ ID NO: 1),  875 LLLLVMADCAFRRNLSTSHTYTTPPY 899  (SEQ ID NO: 2), and  381 GAFASNRTFDIT 392  (SEQ ID NO: 3). 
     In a related aspect, this disclosure relates to a method of producing an EBV antibody using the epitopes disclosed herein according to any known technology. The method entails immunizing an animal such as a mouse or a rabbit with an immunogenic peptide disclosed herein or the epitope represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:3 alone, and screening for and isolating a hybridoma producing an EBV antibody. The EBV antibody produced from such hybridomas can be used for treating or preventing EBV infections. 
     In certain embodiments an antibody for use in the present methods or compositions is a full-length immunoglobulin molecule, which comprises two heavy chains and two light chains, with each heavy and light chain containing three complementary determining regions (CDRs). The CDRs of various antibodies are identified and listed in Table 1 below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 CDR Sequences 
               
            
           
           
               
               
               
            
               
                 Anti- 
                 VH 
                 VL 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 bodies 
                 CDR-1 
                 CDR-2 
                 CDR-3 
                 CDR-1 
                 CDR-2 
                 CDR-3 
               
               
                   
               
               
                 72A1 
                 GSSFTDY 
                 INPYNGG 
                 GGLRRVNWFAYW 
                 TGAVTTSNY 
                 GTN 
                 VLWHSNHWV 
               
               
                   
                 (SEQ ID NO: 4) 
                 (SEQ ID NO: 20) 
                 (SEQ ID NO: 36) 
                 (SEQ ID NO: 52) 
                 (SEQ ID NO: 68) 
                 (SEQ ID NO: 84) 
               
               
                   
               
               
                 HB-1 
                 GFLLTTY 
                 IWAGGS 
                 RDRGYGYLYAMDYW 
                 QNVGTN 
                 STD 
                 QQYNTYPYT 
               
               
                   
                 (SEQ ID NO: 5) 
                 (SEQ ID NO: 21) 
                 (SEQ ID NO: 37) 
                 (SEQ ID NO: 53) 
                 (SEQ ID NO: 69) 
                 (SEQ ID NO: 85) 
               
               
                   
               
               
                 HB-2 
                 GYTFTAY 
                 INYKTGE 
                 PYGYALDYW 
                 SSVNY* 
                 ATS* 
                 QQWSSNPPT* 
               
               
                   
                 (SEQ ID NO: 6) 
                 (SEQ ID NO: 22) 
                 (SEQ ID NO: 38) 
                 (SEQ ID NO: 54) 
                 (SEQ ID NO: 70) 
                 (SEQ ID NO: 86) 
               
               
                   
               
               
                 HB-3 
                 GYTFASY 
                 INPNNGH* 
                 RNLYYYGRPDYW* 
                 QDIGNY* 
                 YTS* 
                 QQGNTLPPT* 
               
               
                   
                 (SEQ ID NO: 7) 
                 (SEQ ID NO: 23) 
                 (SEQ ID NO: 39) 
                 (SEQ ID NO: 55) 
                 (SEQ ID NO: 71) 
                 (SEQ ID NO: 87) 
               
               
                   
               
               
                 HB-5 
                 GYTFTNH 
                 INPYNDY 
                 RSEGWLRRGAWFAY 
                 QSIGTS 
                 YAS 
                 QQSNSWPMLT 
               
               
                   
                 (SEQ ID NO: 8) 
                 (SEQ ID NO: 24) 
                 (SEQ ID NO: 40) 
                 (SEQ ID NO: 56) 
                 (SEQ ID NO: 72) 
                 (SEQ ID NO: 88) 
               
               
                   
               
               
                 HB-6 
                 GYTFTDY* 
                 INTRTGE 
                 PYGYALDYW 
                 SSVNY* 
                 ATS* 
                 QQWSSNPPT* 
               
               
                   
                 (SEQ ID NO: 9) 
                 (SEQ ID NO: 25) 
                 (SEQ ID NO: 41) 
                 (SEQ ID NO: 57) 
                 (SEQ ID NO: 73) 
                 (SEQ ID NO: 89) 
               
               
                   
               
               
                 HB-7 
                 GYTFTDY* 
                 ISPGRSG 
                 RYGHPSYLDVW 
                 QSVGNA 
                 SAS 
                 QQYSSYPLT 
               
               
                   
                 (SEQ ID NO: 10) 
                 (SEQ ID NO: 26) 
                 (SEQ ID NO: 42) 
                 (SEQ ID NO: 58) 
                 (SEQ ID NO: 74) 
                 (SEQ ID NO: 90) 
               
               
                   
               
               
                 HB-8 
                 GYSFTNY* 
                 INTYTGE 
                 RYYYGSVYSAWFAYW 
                 QSIVHSNGNTY* 
                 KVS* 
                 FQGSHVPYT* 
               
               
                   
                 (SEQ ID NO: 11) 
                 (SEQ ID NO: 27) 
                 (SEQ ID NO: 43) 
                 (SEQ ID NO: 59) 
                 (SEQ ID NO: 75) 
                 (SEQ ID NO: 91) 
               
               
                   
               
               
                 HB-9 
                 GFTFSSY 
                 ISSGGSY 
                 REDFYYGSSYGFFDVW 
                 QSIVHSNGNTY* 
                 KVS* 
                 FQGSHVPYT* 
               
               
                   
                 (SEQ ID NO: 12) 
                 (SEQ ID NO: 28) 
                 (SEQ ID NO: 44) 
                 (SEQ ID NO: 60) 
                 (SEQ ID NO: 76) 
                 (SEQ ID NO: 92) 
               
               
                   
               
               
                 HB-10 
                 GYTFTSY* 
                 INPSNGH 
                 RNLYYYGRPDYW 
                 QDIGNY* 
                 YTS* 
                 QQNTLPPT 
               
               
                   
                 (SEQ ID NO: 13) 
                 (SEQ ID NO: 29) 
                 (SEQ ID NO: 45) 
                 (SEQ ID NO: 61) 
                 (SEQ ID NO: 77) 
                 (SEQ ID NO: 93) 
               
               
                   
               
               
                 HB-11 
                 GDSITSG 
                 ISYSGS 
                 RGNGGNYDWYFDVW 
                 SSVNF 
                 YIS 
                 QQFTSSPSWT 
               
               
                   
                 (SEQ ID NO: 14) 
                 (SEQ ID NO: 30) 
                 (SEQ ID NO: 46) 
                 (SEQ ID NO: 62) 
                 (SEQ ID NO: 78) 
                 (SEQ ID NO: 94) 
               
               
                   
               
               
                 HB-12 
                 GYTFTNY* 
                 INPNNGH* 
                 RNLYYYGRPDYW* 
                 QSLVHSNGNTY 
                 KVS* 
                 SQSTHVPLT 
               
               
                   
                 (SEQ ID NO: 15) 
                 (SEQ ID NO: 31) 
                 (SEQ ID NO: 47) 
                 (SEQ ID NO: 63) 
                 (SEQ ID NO: 79) 
                 (SEQ ID NO: 95) 
               
               
                   
               
               
                 HB-14 
                 GYTFTDY* 
                 IHPRRGG 
                 RYGYPWYFDVW 
                 QSIVHDNGNTY 
                 KVS* 
                 FQGSHVPPT 
               
               
                   
                 (SEQ ID NO: 16) 
                 (SEQ ID NO: 32) 
                 (SEQ ID NO: 48) 
                 (SEQ ID NO: 64) 
                 (SEQ ID NO: 80) 
                 (SEQ ID NO: 96) 
               
               
                   
               
               
                 HB-17 
                 GYTFTSY* 
                 INPNNGH* 
                 RNLFYYSRPDYW 
                 QDIGNY* 
                 YTS* 
                 QQGNTLPPT* 
               
               
                   
                 (SEQ ID NO: 17) 
                 (SEQ ID NO: 33) 
                 (SEQ ID NO: 49) 
                 (SEQ ID NO: 65) 
                 (SEQ ID NO: 81) 
                 (SEQ ID NO: 97) 
               
               
                   
               
               
                 HB-20 
                 GYTFTSY* 
                 INPTNGH 
                 RNLYYYGRPDYW* 
                 QDIGNY* 
                 YTS* 
                 QQGNALPPT 
               
               
                   
                 (SEQ ID NO: 18) 
                 (SEQ ID NO: 34) 
                 (SEQ ID NO: 50) 
                 (SEQ ID NO: 66) 
                 (SEQ ID NO: 82) 
                 (SEQ ID NO: 98) 
               
               
                   
               
               
                 HB-22 
                 GFSLTNY 
                 IWSDGS 
                 RNYYGNSYPAWFAYW 
                 QSIVHSNGNTY 
                 KVS* 
                 FQGSHVPWT 
               
               
                   
                 (SEQ ID NO: 19) 
                 (SEQ ID NO: 35) 
                 (SEQ ID NO: 51) 
                 (SEQ ID NO: 67) 
                 (SEQ ID NO: 83) 
                 (SEQ ID NO: 99) 
               
               
                   
               
            
           
         
       
     
     The term “antibody,” in addition to natural antibodies, also includes genetically engineered or otherwise modified forms of immunoglobulins, such as synthetic antibodies, intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, peptibodies and heteroconjugate antibodies (e.g., bispecific antibodies, multispecific antibodies, dual-specific antibodies, anti-idiotypic antibodies, diabodies, triabodies, and tetrabodies). The antibodies disclosed herein can be monoclonal antibodies or polyclonal antibodies. In those embodiments wherein an antibody is an immunologically active portion of an immunoglobulin molecule, the antibody may be, for example, a Fab, Fab′, Fv, Fab′ F(ab′) 2 , disulfide-linked Fv, single chain Fv antibody (scFv), single domain antibody (dAb), or diabody. The antibodies disclosed herein, including those that are immunologically active portion of an immunoglobulin molecule, retain the ability to bind a specific antigen, for example, EBV gp350 or one or more epitopes thereof, EBV gp220 or one or more epitopes thereof, or to bind a specific fragment of gp350/gp220. 
     In some embodiments, the EBV antibodies disclosed herein have undergone post-translational modifications such as phosphorylation, methylation, acetylation, ubiquitination, nitrosylation, glycosylation, or lipidation associated with expression in a mammalian cell line, including a human or a non-human host cell. Techniques for producing recombinant antibodies and for in vitro and in vivo modifications of recombinant antibodies are known in the art. 
     Provided in certain embodiments herein are chimeric, and/or humanized EBV antibodies. Various techniques are known in the art for humanizing antibodies from non-human species such that the antibodies are modified to increase their similarity to antibodies naturally occurring in humans. Six CDRs are present in each antigen binding domain of a natural antibody. These CDRs are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration. CDR sequences of certain antibodies identified herein are shown in Table 1. The remainder of the amino acids in the antigen binding domains, referred to as “framework” regions, show less inter-molecular variability and form a scaffold to allow correct positioning of the CDRs. This disclosure also relates to antibodies comprising VH and VL regions comprising the CDRs shown in Table 1. 
     “Treating” or “treatment” of a disease or a condition may refer to preventing the disease or condition, slowing the onset or rate of development of the disease or condition, reducing the risk of developing the disease or condition, preventing or delaying the development of symptoms associated with the disease or condition, reducing or ending symptoms associated with the disease or condition, generating a complete or partial regression of the disease or condition, or some combinations thereof. 
     As used herein, the term “subject” refers to mammalian subject, preferably a human. The phrases “subject” and “patient” are used interchangeably herein. 
     The method for treating a condition or a viral infection includes administering a therapeutically effective amount of a therapeutic agent or a pharmaceutical composition. An “effective amount,” “therapeutically effective amount” or “effective dose” is an amount of a composition (e.g., a therapeutic agent or a pharmaceutical composition) that produces a desired therapeutic effect in a subject, such as preventing or treating a target disease or condition or alleviating symptoms associated with the disease or condition. The precise therapeutically effective amount is an amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic agent (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject&#39;s response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy 21 st  Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams &amp; Wilkins, Philadelphia, Pa., 2005. 
     The pharmaceutical composition may include, among other things, an EBV antibody disclosed herein or an immunogenic fragment thereof, one or more gp350 epitopes represented by SEQ ID NOs: 1-3, one or more immunogenic peptides disclosed herein, or an EBV antibody-small molecule conjugate disclosed herein. 
     The pharmaceutical composition may also include one or more pharmaceutically acceptable carriers. A “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. 
     The pharmaceutical compositions described herein may be administered by any suitable route of administration. A route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch), or vaginal. “Transdermal” administration may be accomplished using a topical cream or ointment or by means of a transdermal patch. “Parenteral” refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. In some embodiments, the therapeutic compositions described herein are administered by intravenous injection or intraperitoneal injection. 
     The epitopes disclosed herein have various uses. For example, the epitopes can be used to produce immunogenic peptides. Such an immunogenic peptide comprises one or more gp350 epitopes having an amino acid sequence identical to or sharing at least 60% similarity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the immunogenic peptide comprises a first domain comprising an amino acid sequence having at least 60% similarity to SEQ ID NO:1, a second domain comprising an amino acid sequence having at least 60% similarity to SEQ ID NO: 2, and a third domain comprising an amino acid sequence having at least 60% similarity to SEQ ID NO: 3. In some embodiments, the immunogenic peptide comprises a first domain comprising an amino acid sequence having at least 60% similarity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and a second domain comprising a known immunogenic peptide such as keyhole limpet hemocyanin (KLH) peptide. 
     Additionally, the epitopes or immunogenic peptides disclosed herein can be used for producing anti-gp350 antibodies using any existing technology. 
     In certain embodiments, disclosed herein is a method of treating or preventing EBV infection in a subject, comprising administering a therapeutically effective amount of an anti-gp350 antibody disclosed herein or an immunogenic fragment thereof, one or more epitopes represented by SEQ ID NOs: 1-3, an immunogenic peptide described herein, an EBV antibody-drug conjugate described herein, or a pharmaceutical composition comprising the anti-gp350 antibody or an immunogenic fragment thereof, one or more epitopes, one or more immunogenic peptides, or the EBV antibody-drug conjugate. 
     In certain embodiments, disclosed herein is a method of treating or preventing EBV infection in a subject, comprising administering a therapeutically effective amount of an anti-gp350 antibody disclosed herein or an immunogenic fragment thereof, one or more epitopes represented by SEQ ID NOs: 1-3, an immunogenic peptide described herein, an EBV antibody-drug conjugate described herein, or a pharmaceutical composition comprising the anti-gp350 antibody or an immunogenic fragment thereof, one or more epitopes, one or more immunogenic peptides, or the EBV antibody-drug conjugate. 
     In certain embodiments, disclosed herein is a method of treating or preventing EBV-associated PTLD in a subject, comprising administering a therapeutically effective amount of an anti-gp350 antibody disclosed herein or an immunogenic fragment thereof, one or more epitopes described herein, one or more immunogenic peptides described herein, an EBV antibody-drug conjugate described herein, or a pharmaceutical composition comprising the anti-gp350 antibody or an immunogenic fragment thereof, one or more epitopes, one or more immunogenic peptides, or the EBV antibody-drug conjugate, before or after the transplant in the subject. 
     As shown in the working examples, 15 novel EBV gp350-specific mAbs were generated, their binding to gp350 was characterized, their neutralization activity against EBV infection in vitro was determined, their cognate epitopes were mapped, and the exact aa residues they recognize on gp350 were defined. Six of the eight previously described epitopes responsible for generating neutralizing and non-nAbs were confirmed and the exact aa residues that they bind were defined. This study also confirmed that the binding epitopes on gp350 that elicit nAbs are between aa 4-443 (59). An additional neutralizing epitope and two new non-neutralizing epitopes, with one located downstream of the gp350 ectodomain (aa 1-841) were identified. Importantly, the newly developed mAbs have many uses in vaccine development, diagnosis of viral infection, and therapeutic/prophylactic management of post-transplant lymphoproliferative diseases, either individually, in combination with nAb-72A1, or with other mAbs such as anti-gH/gL (E1D1). 
     To overcome the existing challenges facing PTLD treatment, novel EBV antibodies and EBV antibody-drug conjugates (ADCs) are developed. The EBV neutralizing antibodies (nAbs) that specifically block new or reactivated EBV infection are conjugated with small molecules that specifically target latent viral protein, EBV nuclear antigen 1 expressed in all EBV+ malignancies. The recent identification and isolation of nAbs against the highly variable viruses HIV-1 (10, 11), influenza (12-14), and respiratory syncytial virus (15) has direct implications for successful EBV protection. Indeed, in 2012, an international, multidisciplinary expert panel recommended use of intravenous (IV) anti-viral nAbs for preventing or treating EBV+ PTLD (16). EBV uses multiple surface glycoproteins (gps), including the major gp350, to infect host cells (17, 18). These gps are expressed on EBV virions and in EBV+ cells (19, 20), and stimulate immune responses in humans and in animal models (21-23), making them attractive targets for an EBV vaccine (24). Multiple lines of evidence suggest that use of anti-gp350 nAbs to protect against EBV-PTLDs is feasible (16): (A) Maternal Abs protect against EBV infection in neonates (25, 26); (B) gp350-expressing EBV+ cells activate complement (27) and mediate Ab-dependent cellular cytotoxicity (28); (C) gp350 vaccines reduce EBV load and protect against EBV+ lymphomas in marmosets (29-32) and protect EBV-naive adults from EBV-induced mononucleosis (32-34); (D) Compared to control mice, SCID mice injected with peripheral blood mononuclear cells from EBV-naive donors and immunized with anti-gp350 (72A1) mouse nAb are completely protected against EBV and development of EBV+ tumors or PTLD-like lesions (35); and (E) 72A1 also conferred short-term protection against acquiring EBV after transplantation in 3 out of 4 pediatric patients in a small phase 1 clinical trial (35). However, there was a major drawback: all 4 patients who received 72A1 developed human anti-mouse Abs (HAMA), which can cause side effects and limit treatment efficacy, with one developing a hypersensitivity reaction. This suggests that 72A1 in its native form is not a safe treatment for humans (35). Thus, chimeric (human/mouse), humanized, or human nAbs, which are safe and effective in the treatment of various cancers, are needed (7, 36-38). 
     Pre-existing antibodies provide the primary defense against viral infection. Prophylactic prevention of EBV primary infection has mainly focused on blocking the first step of viral entry by generating neutralizing antibodies (nAbs) that target EBV envelope glycoproteins. Five glycoproteins, in particular, gp350/220 (gp350), gp42, gH, gL, and gB, are required for efficient infection of permissible host cells and have emerged as potential prophylactic targets (23, 24, 61, 64). 
     Several studies have indicated that the EBV gp350 as the major immunodominant glycoprotein is an ideal target for EBV nAbs production. Although the ectodomain of EBV gp350 (aa 1-841) has been shown to contains at least eight unique CD21 binding epitopes, only one of these epitopes (aa 142-161) is capable of eliciting nAbs (57-58). The aa residues that constitute the other epitopes and their role in generating nAb has not been elucidated, as this information would be valuable in the precise design of effective EBV peptide vaccine. To date, nAb-72A1 remains the only EBV antibody with proven clinical prophylactic efficacy, as its been shown to confer short-term protection by reducing and delaying EBV infection onset in immunized pediatric transplant patients (35). 
     EBV predominantly infects epithelial cells and B cells, reflecting the viral tropism and the cellular ontogeny for EBV-associated malignancies (17). There are two schools of thought on how the initial EBV transmission into the human host occurs. In the first infection model, the incoming virus engages with ephrin receptor A2 via heterodimeric gH/gL, which triggers gB fusion with the epithelial cell membrane and entry of the virus into the cytoplasm (17). This interaction is thought to occur in the oral mucosa, where the virus undergoes lytic replication to release virions that subsequently infect B cells. In the alternative model, the incoming virus binds to the host cell via complement receptor type 1 (CR1)/CD35 (45) and/or CR2/CD21 through its major immunodominant glycoprotein, gp350 (65). The interaction between gp350 and CD35 and/or CD21 triggers viral adsorption, capping, and endocytosis into the B cell (66), which subsequently leads to interaction between heterotrimeric viral glycoproteins complex, gp42/gH/gL, binding to HLA class II molecules to activate gB membrane fusion and entry. Because these two models are not necessarily mutually exclusive, and given that both gp350 and gH/gL complex are important in initiating the first viral contact with host cells, use of nAbs that target either gp350 or gH/gL complex or both may potently block incoming virus at the oral mucosa. 
     Nearly all EBV-infected individuals develop nAbs directed to the ectodomains of these glycoproteins (52, 67). These antibodies can prevent neonatal infection, and can protect against acute infectious mononucleosis in adolescents and several human lymphoid and epithelial malignancies associated with EBV infection (30, 32-34). Although numerous monoclonal antibodies (mAbs) have been generated against EBV gp350 (53, 68, 69), only two murine mAbs, the non-neutralizing 2L10 and the neutralizing 72A1, have been extensively characterized and made commercially available (68, 69). Importantly, nAb-72A1 conferred short-term clinical protection against EBV transmission after transplantation in pediatric patients in a small phase I clinical trial (35). 
     EBV gp350 is the most immunogenic envelope glycoproteins on the virions. It is a type 1 membrane protein that encodes for 907 amino acid (aa) residues. A single splice of the primary transcript deletes 197 codons and joins gp350 codons 501 and 699, in frame, to generate the gp220 messenger RNA. Both gp350 and gp220 are comprised of the same 18-aa residue at the C terminus that is located within the viral membrane, a 25-aa residue at the transmembrane-spanning domain, and a large highly glycosylated N-terminal ectodomain, aa 1-841 (57). The first 470 aa of gp350 are sufficient for binding CD21 in B cells, as demonstrated by a truncated gp350 (aa 1-470) blocking the binding of EBV to B cells and reducing viral infectivity (17). The gp350-binding domain on CD21 is mapped to N-terminal short consensus repeats (SCRs) 1 and 2, which also bind to a bioactive fragment of complement protein 3 (C3d) (34, 68). A soluble truncated EBV gp350 fragment (aa 1-470) and soluble CD21 SCR1 and SCR2 can block EBV infection and immortalization of primary B cells (57). However, gp350 binding to CD35 is not restricted to N-terminal SCRs; it binds long homologous repeat regions as well as SCRs 29-30 (57). 
     The gp350 ectodomain is heavily glycosylated, with both N- and O-linked sugars, which accounts for over half of the molecular mass of the protein. Currently there is only one crystal structure available for gp350, comprised of a truncated structure between 4-443 aa, with at least 14 glycosylated arginines coating the protein with sugars, with the exception of a single glycan-free patch (59). Mutational studies of several residues in the glycan-free patch resulted in the loss of CD21 binding (59), suggesting that binding of CD35 and CD21 by gp350 is mediated within this region. 
     There are at least eight unique CD21 binding epitopes located at the N-terminus of the gp350 ectodomain (58); at least one of these epitopes (aa 142-161) is capable of eliciting nAbs (57, 58). The aa residues 142-161 are also the binding site for nAb-72A1 (59, 68). Using gp350 synthetic peptides binding to CD21 on the surface of a B cell line, additional gp350 epitope was identified in the C-terminal region of gp350 (aa 822-841), suggesting it is involved in EBV invasion of B cells (58). The role of other epitopes in eliciting nAbs has not been fully investigated. Furthermore, the exact aa residues that comprise the core binding sites for epitope capable of eliciting neutralization and non-neutralization antibodies have not been determined. Mapping the EBV gp350 protein residues defining immunodominant epitopes, identifying the critical aa residues of the eight epitopes, and defining their roles in generating neutralizing and non-nAbs will guide rational design and construction of an efficacious EBV gp350-based vaccine that would focus the B-cell responses to the protective epitopes. 
     As demonstrated in the working examples, 23 hybridomas producing antibodies against EBV gp350 were generated. To assess their clinical potential and utility in informing future prophylactic and therapeutic vaccine design: (1) the ability of the antibodies produced by the new hybridomas to detect gp350 protein was tested by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblot; (2) the unique CDRs of the heavy and light chains of all 23 hybridomas were sequenced to identify novel mAbs; (3) the efficacy of each mAb to neutralize EBV infection in vitro was measured; and (4) RepliTope peptide microarrays were used to identify gp350 core aa residues recognized by neutralizing and non-neutralizing mAbs. Using the newly generated antibodies, a new epitope bound preferentially by nAbs was identified, distinct from the canonical neutralizing epitope bound by nAb-72A1, as well as two immunodominant epitopes bound by both neutralizing and non-nAbs. 
       FIG.  1    demonstrates characterization of the 23 gp350-specific antibodies produced by hybridomas and their ability to bind gp350 protein.  FIG.  2    demonstrates that out of 23 hybridomas, 15 hybridomas were determined to be monoclonal and novel based on the VH and VL CDR sequences compared to those of the reported nAb-72A1 (57). 
     Following confirmation that the new mAbs recognized EBV gp350 antigen and had unique VH-VL sequences, further characterization revealed that mAbs HB9, HB10, HB11, HB17, and HB20 inhibited EBV infection in a dose-dependent manner, with HB17 and HB20 being the best neutralizers ( FIG.  3   ). Thus, provided herein are new nAbs and non-nAbs against EBV infection for therapeutic and prophylactic uses. 
     Various methods, including lectin/ricin immune-affinity assay (69), purified mAbs (53, 70), purified soluble gp350 mutants (57), synthetic peptides (51, 55, 54), cell binding assays (58), and crystal structure of partial gp350 protein (4-443) (59), have been used to identify the critical gp350 epitopes responsible for its interaction with the CD35 and CD21 cellular receptors (for a detailed summary review see Table 2). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Summary of gp350 Epitope Mapping over Time Using Different Methodologies 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Number of 
                   
               
               
                 Method 
                 mAbs/protein/peptides 
                 epitopes 
                 Reference 
               
               
                   
               
               
                 Competitive binding assay 
                 Newly generated mAbs from 
                 8 epitopes- 
                 Qualtiere et 
               
               
                 Tagged mAb vs untagged mAbs 
                 purified virus or whole cell 
                 Sequence not 
                 al., 1987 (53) 
               
               
                   
                 perp after induction 
                 defined 
               
               
                 Binding studies- 
                 Newly generated mAbs (61) 
                 2 possible regions 
                 Nemerow et 
               
               
                 determine the effects of 
                   
                 identified by sequence 
                 al 1987 (54) 
               
               
                 anti-gp350 mAbs on gp350 
                   
                 alignment to C3d sequence. 
               
               
                 binding to CR2 
                   
                 1. aa 21-28 
               
               
                   
                   
                 2. aa 372-378 
               
               
                 Binding studies 
                 Peptide and protein 
                 1. aa 21-28 
                 Nemerow et 
               
               
                   
                   
                 2. N-terminus of gp350 
                 al 1989 (55) 
               
               
                 Dot Blot immunoasaay 
                 Protein - 8 clones 
                 3 sequences defined 
                 Zhang et 
               
               
                 Purified truncated protein 
                 overlapping N and C terminus 
                 1. aa 310-325 
                 al., 1991 (56) 
               
               
                 incubated with mAbs 
                 portions of protein mAbs from 
                 2. aa 326-439 
               
               
                   
                 Qualtiere et al., 1987 
                 3. aa 733-841 
               
               
                 Peptide digest and 
                 Truncated and 
                 Narrowed down to the 
                 Tanner et 
               
               
                 immunoprecipitation 
                 mutant protein mAbs 
                 first 470 residues 
                 al., 1998 (57) 
               
               
                   
                 (72A1 and BOS-1) 
               
               
                 Peptide cell binding assay- 
                 Synthesized peptides 
                 7 regions 3 identified 
                 Urquiza et 
               
               
                 to 2 CR2 positive 
                 covering gp350 (907aa) 
                 1. aa 142-161 
                 al 2005 (58) 
               
               
                 (Raji and Ramos) and 1 
                   
                 2. aa 282-301 
               
               
                 negative (P3HR-1) cell line 
                   
                 3. aa 822-841 
               
               
                 Crystal structure 
                 Mutant proteins, mAbs 72a1 
                 3 epitopes (based 
                 Szakonyi et 
               
               
                 and binding studies 
                   
                 on 72A1 binding 
                 al., 2006 (59) 
               
               
                   
                   
                 and gp350 4-443) 
               
               
                   
                   
                 1. aa 16-29 
               
               
                   
                   
                 2. aa 142-161 
               
               
                   
                   
                 3. aa 282-301 
               
               
                 Structural docking studied 
                 Gp350 and CR2 crystal 
                 Single epitope 
                 Sitompul et 
               
               
                 and antigenicity mapping 
                 structure alignment/docking 
                 (based on gp350 
                 al., 2012 (60) 
               
               
                   
                   
                 (1-470)) 
               
               
                   
                   
                 1. aa147-165 
               
               
                 Structural alignment 
                 Peptides 
                 3 epitopes 
                 Tanner et 
               
               
                 (computer modeling of gp350 
                 (used in immunization) 
                 1. aa 14-20 
                 al., 2015 (51) 
               
               
                 and 72A1)/docking studies 
                 and mAb (72A1) 
                 2. aa 144-161 
               
               
                   
                   
                 3. aa 194-211 
               
               
                   
               
            
           
         
       
     
     The epitope mapping assay identified a total of nine epitopes including two new epitopes, one bound by all mAbs, including nAb-72A1 and 2L10, regardless of the mAb neutralizing capability suggesting it is an immunodominant epitope on the gp350 protein. Portions of six of the previously described epitopes, including the only currently recognized neutralizing epitope were also identified and their exact aa residues were defined. However, two previously reported epitopes located at aa 282-301 and aa 194-211, which have been reported to be involved in the binding of nAb-72A1 or CD21, respectively (51, 58, 59), were not identified. 
     As demonstrated in the working examples, the comparative epitope mapping analysis results identified a novel neutralizing epitope,  381 GAFASNRTFDIT 392  (SEQ ID NO; 3), which was bound preferentially by the nAbs HB20 and nAb-72A1, but not the non-neutralizing mAb HB5. The new neutralizing epitope is distinct from the reported canonical nAb-72A1 binding epitope aa 142-161, on gp350, suggesting that epitope 381-392 is a novel epitope on gp350 capable of eliciting nAbs using existing technology and protocols. 
     Virus-specific treatments are less likely to target basic metabolic mechanisms of healthy cells, making them more likely to efficiently kill virus-infected cells with fewer side effects. Until recently, few drug regimens have specifically targeted EBV+ lymphomas. However, in 2015, a few small molecules showed activity against EBV-transformed cells (39). Furthermore, in 2017, Jiang et al. described a novel small molecule (L2P4) that shows discriminating anti-proliferative activities against EBV-transformed B lymphoma cells (40). 
     Having described the invention with reference to the embodiments and illustrative examples, those in the art may appreciate modifications to the invention as described and illustrated that do not depart from the spirit and scope of the invention as disclosed in the specification. The examples are set forth to aid in understanding the invention but are not intended to, and should not be construed to limit its scope in any way. The examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art and are described in numerous publications. Further, all references cited above and in the examples below are hereby incorporated by reference in their entirety, as if fully set forth herein. 
     EXAMPLES 
     Example 1: Materials and Methods 
     Cells and viruses. EBV-AGS, a human gastric carcinoma cell line infected with a recombinant Akata virus expressing enhanced fluorescent green protein (eGFP) was a kind gift of Dr. Lisa Selin (University of Massachusetts Medical School). Anti-EBV gH/gL (E1D1) hybridoma cell line was a kind gift of Dr. Lindsey Hutt-Fletcher (Louisiana State University Health Sciences Center). Chinese hamster ovary cells (CHO); human embryonic kidney cells expressing SV-40 T antigen (HEK-293T); HEK-293 6E suspension cells; EBV-positive Burkitt lymphoma cells (Raji); myeloma cells (P3X63Ag8.653); and anti-EBV gp350 nAb-72A1 hybridoma cells (HB168) were purchased from American Type Culture Collection (ATCC). EBV-AGS cells were maintained in Ham&#39;s F-12 media supplemented with 500 μg/ml neomycin (G418, Gibco). Raji, P3X63Ag8.653, and HB168 hybridoma cells were maintained in RPMI 1640. CHO and HEK-293T cells were maintained in DMEM. HEK-293 6E cells were maintained in FreeStyle F17 Expression Medium supplemented with 0.1% Pluronic F-68. All culture media were supplemented with 10% fetal bovine serum (FBS), 2% penicillin-streptomycin, and 1% I-glutamine, with the exception of Freestyle F17 expression medium. 
     Antibodies and plasmids. Primary antibodies: EBV anti-gp350 nAb (72A1) and anti-gH/gL (E1D1) were purified from the supernatant of HB168 and E1D1 hybridoma cell lines, respectively, using Capturem™ Protein A Maxiprep spin columns (Takara). Anti-gp350/220 mAb (2L10) was purchased from Millipore Sigma. 
     Secondary antibodies: Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for immunoblot or ELISA were purchased from Bio-Rad. Alexa Fluor (AF) 488-conjugated goat anti-mouse IgG (H+L) for flow cytometry was purchased from Life Sciences Tech. Goat anti-mouse IgG (H+L) secondary antibody and DyLight 650 for epitope mapping were purchased from Thermo Fisher Scientific. 
     The construction of the pCI-puro vector and pCAGGS-gp350/220-F has been described (23, 45). 
     Virus production and purification. eGFP-tagged EBV was produced from the EBV-infected AGS cell line as described (46). Briefly, EBV-AGS cells were seeded to 90% confluency in T-75 flasks in Ham&#39;s F-12 medium containing G418 antibiotic. After the cells reached confluency, G418 media was replaced with Ham&#39;s F-12 medium containing 33 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3 mM sodium butyrate (NaB) to induce lytic replication of the virus. Twenty-four h post-induction, the media was replaced with complete Ham&#39;s F-12 media without G418, TPA, or NaB and cells were incubated for 4 days at 37° C. The cell supernatant was collected, centrifuged, and filtered using 0.8 μm filter to remove cell debris. The filtered supernatant was ultra-centrifuged using a Beckman-Coulter type 19 rotor for 70 min at 10,000 rpm to pellet the virus. EBV-eGFP virus was titrated in both HEK-293T cells and Raji cells, and stocks were stored at −80° C. for subsequent experiments. 
     Generation and purification of gp350 virus-like particles. To generate gp350 VLPs, equal amounts (8 μg/plasmid) of the relevant plasmids (pCAGGS-Newcastle disease virus (NDV) M, —NP, and gp350 ectodomain fused to fusion protein cytoplasmic and transmembrane domains) were co-transfected into 80% confluent CHO cells seeded in T-175 cm2 flasks; supernatant from transfected cells was collected and VLPs were purified and composition characterized as previously described (47). 
     Production of hybridoma cell lines. Seven days prior to immunization, two eight-week-old BALB/c mice were bled for collection of pre-immune serum. The mice were immunized with purified UV-inactivated EBV three times (Day 0, 21, and 35), and boosted every 7 days three times (Day 42, 49, and 56) with VLPs incorporating gp350 on the surface after Day 35. The mice were sacrificed, and their splenocytes were isolated, purified, and used to fuse with P3X63Ag8.653 myeloma cells at a ratio of 3:1 in the presence of polyethylene glycol (PEG, Sigma). Hybridoma cells were seeded in flat-bottom 96-well plates and selected in specialized hybridoma growth media with HAT (Sigma) and 10% FBS. 
     Indirect ELISA. Hybridoma cell culture supernatant from wells that had colony-forming cells were tested for antibody production by indirect ELISA. Briefly, immunoplates (Costar 3590; Corning Incorporated) were coated with 50 μl of 0.5 μg/ml recombinant EBV gp350/220 (Millipore) protein diluted in phosphate buffered saline (PBS, pH 7.4) and incubated overnight at 4° C. After washing three times with PBS containing 0.05% (v/v) Tween 20 (washing buffer), plates were blocked with 100 μl washing buffer containing 2% (w/v) bovine serum albumin (BSA) then incubated for 1 h at room temperature and washed as above. 100 μl of hybridoma supernatant was added to each well (in triplicate) and incubated for 2 h at room temperature. PBS and nAb-72A1 were added as negative and positive controls, respectively. The plates were washed as described above, followed by incubation with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:2,000 diluted in PBS) at room temperature for 1 h. The plates were washed again and the chromogenic substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, Life Science Technologies) was added. The reaction was stopped using ABTS peroxidase stop solution containing 5% sodium dodecyl sulfate (SDS) in water. The absorbance was read at an optical density of 405 nm using an ELISA reader (Molecular Devices). Three independent were performed. 
     Antibody purification, quantification, and isotyping. Hybridoma cells from selected individual positive clones were expanded stepwise from 96-well plates to T-75 flasks. At confluence in T-75 flasks, supernatant from individual clones was collected, clarified by centrifugation (4,000 g, 10 min, 4° C.), and filtered through a 0.22-μm-membrane filter (Millipore). Antibodies were further purified by Capturem™ Protein A Maxiprep (Takara) and stored in PBS (pH 7.4) at 4° C. Antibodies were analyzed by SDS-PAGE to determine purity. Bicinchoninic acid assay (BCA assay; Thermo Fisher Scientific) was conducted to determine the concentration of purified antibodies. Isotype identification was performed with the Rapid ELISA mouse mAb isotyping kit (Thermo Fisher Scientific). 
     RNA extraction, cDNA synthesis, and sequencing of the variable region of the mAbs. Total RNA was extracted from 1×10 6  hybridoma cells using the RNeasy Mini Kit (Qiagen). Each hybridoma clone cDNA was synthesized in a total volume of 20 μl using Tetro Reverse Transcriptase (200 u), RiboSafe RNase Inhibitor, Oligo(dT)18 primer, dNTP mix (10 mM each nucleotide), and 100-200 ng RNA. Reverse transcription was performed at 45° C. for 30 min, and terminated at 85° C. for 5 min. The cDNA was stored at −20° C. Immunoglobulin (Ig) VH and VL were amplified using the mouse Ig specific primer set purchased from Novagen (48). The VH and VL genes were amplified in separate reactions and PCR products were visualized on 1% agarose gel. 
     The VH and VL amplicons were sequenced using an Illumina MiSeq platform: duplicate 50 μl PCR reactions were performed, each containing 50 ng of purified cDNA, 0.2 mM dNTPs, 1.5 mM MgCl2, 1.25 U Platinum Taq DNA polymerase, 2.5 μl of 10×PCR buffer, and 0.5 μM of each primer designed to amplify the VH and VL. The amplicons were purified using AxyPrep Mag PCR Clean-up kit (Thermo Fisher Scientific). The Illumina primer PCR PE1.0 and index primers were used to allow multiplexing of samples. The library was quantified using ViiA™ 7 Real-Time PCR System (Life Technologies) and visualized for size validation on an Agilent 2100 Bioanalyzer (Agilent Technologies) using a high-sensitivity cDNA assay. The sequencing library pool was diluted to 4 nM and run on a MiSeq desktop sequencer (Illumina). The 600-cycle MiSeq Reagent Kit (Illumina) was used to run the 6 μM library with 20% PhiX (Illumina), and FASTQ files were used for data analysis. 
     Chimeric mAb construct generation. To generate chimeric mAbs, the VH and VL sequences were cloned into the dual-vector system pFUSE CHIg/pFUSE CLIg (InvivoGen), which express the constant region of the heavy and light chains of human immunoglobulins, respectively (Genewiz). The constructs were transiently transfected into HEK-293 6E cells. The supernatants were collected at 72 h post-transfection and IgG was purified using protein A/G affinity chromatography. 
     Immunoblot analysis. CHO cells were cultured and stably co-transfected with pCAGGS-gp350/220 F and pCI-puro vector containing a puromycin resistance gene. Forty-eight h post-transfection, DMEM media containing 10 μg/ml of puromycin was added to enrich for cells expressing gp350 protein. Puromycin-resistant clones were expanded, followed by flow cytometry sorting using nAb-72A1 to a purity &gt;90%. EBV gp350-positive CHO cells were harvested and lysed in radioimmunoprecipitation assay buffer (RIPA) followed by centrifugation at 15,000 g for 15 min on a benchtop centrifuge. The supernatants were collected and heated at 95° C. for 10 min in SDS sample buffer containing β-mercaptoethanol, then separated using SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane using an iBlot™ Transfer System (Thermo Fisher Scientific) followed by incubation in blocking buffer (1% BSA; 20 mM Tris-HCl, pH 7.5; 137 mM NaCl; and 0.1% Tween-20 [TBST]) for 1 h. The blots were incubated in TBST containing purified anti-gp350 antibodies (1:50) overnight at 4° C. After three washes with TBST, the blots were incubated with a goat anti-mouse conjugated to horseradish peroxidase (1:2000) in TBST for 1 h. After three washes, the antibody-protein complexes were detected using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). All experiments were independently repeated three times. 
     Flow cytometry. To assess the ability of purified anti-gp350 mAb to detect surface expression of EBV gp350 protein by flow cytometry, CHO cells that stably express EBV gp350 were harvested and stained with purified anti-gp350 (10 μg/ml), followed by Alexa Fluor® 488 goat anti-mouse IgG secondary antibody. Flow cytometric analysis was performed on a C-6 FC (BD Biosciences) and data was analyzed using FlowJo Cytometry Analysis software (FlowJo, LLC) as described (47). All the experiments were independently repeated three times. 
     EBV neutralization assay. Purified individual anti-gp350 mAbs were incubated with purified AGS-EBV-eGFP (titer calculated to infect at least 20% of HEK293 cells seeded in 100 μl of serum-free DMEM) for 2 h at 37° C. To represent EBV infection of B cells, the pre-incubated anti-gp350 mAbs/AGS-EBV-eGFP were used to infect 5×105 Raji cells seeded in a 96-well plate. Anti-gp350 neutralizing 72A1 and non-neutralizing 2L10 mAbs served as positive and negative controls, respectively. Plates were incubated at 37° C. and the number of eGFP+ cells was determined using flow cytometry 48 h post-infection. All dilutions were performed in quintuplicate and the assays repeated three times for Raji cells. Antibody EBV neutralization activity was calculated as: % neutralization=(EBValone−EBVmAb)/(EBValone)×100. 
     Epitope mapping. Anti-gp350 mAbs were incubated with a multi-well EBV GP350/GP340 RepliTope (JPT) peptide microarray displaying 224 peptides (15mers with 11 aa overlap) in 3×7 subarrays. Briefly, anti-gp350 mAbs were diluted in blocking solution (TBS-T and 2% BSA) to a final concentration of 10 μg/ml and incubated with the microarray slide for 1 h at 30° C. with shaking. Slides was washed 5 times with wash buffer (TBS-T), followed by incubation with 1 μg/ml secondary antibody Dylight 650 (Thermo Fisher Scientific) for 1 h at 30° C. After washing 5 times with wash buffer and 2 times with distilled water, microarray slides were dried by centrifugation. Detection was performed using the Agilent DNA microarray scanner. 
     Example 2: Development and Characterization of Monoclonal Antibodies Against EBV Gp350 
     New EBV gp350-specific mAbs were generated and biochemically characterized, and their ability to neutralize EBV infection was evaluated. In addition, the antibodies were used to map immunodominant epitopes on the EBV gp350 protein. 23 novel monoclonal antibodies specific against EBV gp350 were developed. To generate hybridomas, BALB/c mice were immunized with purified UV-inactivated EBV and boosted with virus-like particles (VLPs) that incorporate EBV gp350 ectodomain on the surface to enrich for production of anti-gp350 antibodies, then splenocytes were isolated from the immunized mice and fused with myeloma cells. Specifically, five eight-week-old BALB/c mice were immunized with virus-like particles incorporating gp350/220 on the surface, four times (day 0, 14, 28, and 56) via intraperitoneal injection without adjuvants. At day 64, immunized mice were boosted once intravenously. Animals were sacrificed at Day 70 to harvest splenocytes for fusion with the mouse myeloma P3X63Ag8 cell line. 
     Indirect ELISA was used to screen supernatants from the hybridomas for specificity against purified EBV gp350 ectodomain protein (aa 4-863) and 23 hybridomas producing gp350 specific antibodies were identified. These novel antibodies were analyzed by flow cytometric analysis for surface expression of gp350 protein on 10 6  CHO cells transfected with 1 μg of pCAGGS-gp350. gp350 expressing cells were resuspended in PBS, stained with anti-gp350 mAb, which detects the gp350 ED, followed by secondary Ab goat anti-mouse conjugated to AF488. Additionally, western blot analysis was conducted on untransfected and pCAGGS-gp350 transfected CHO lysate. Anti-gp350 mAb 72A1 was used as a positive control (1:100) and anti-gp350 hybridoma clone supernatants were used at 1:50, and anti-mouse secondary antibody was used at 1:2000. 
     The isotypes of the new antibodies were determined to be IgG1 (n=14), IgG2a (n=5), IgG2b (n=1), a mixture of IgG1 and IgG2b (n=1), and a mixture of IgG1 and IgM (n=2). It was found that all 23 hybridoma producing antibodies, designated HB1-23, recognized the gp350 antigen in an initial ELISA screening using unfractionated and unpurified hybridoma supernatants (data not shown). Affinity purification with protein A followed by SDS-PAGE was used to confirm the purity of the antibodies. When quantified amount of the purified antibodies (10 μg/ml) was re-evaluated using indirect ELISA, all of the 23 antibodies had ELISA signals two times greater than those of phosphate buffered saline (negative control), and were considered as positive or specific to gp350. Of these, five (HB4, HB5, HB7, HB13, and HB14) demonstrated binding affinity equal to or greater than that of the positive control, nAb-72A1 ( FIG.  1 A ). This difference in binding of the 23 antibodies could be due to differential exposure of cognate epitopes on gp350 in the assay performed. 
     Determining the nature of the binding between an antibody and its target antigen is an important consideration for the performance and specificity of an antibody, as it can involve the recognition of a linear or conformational epitope (49). The antibodies were characterized using immunoblot analysis of denatured gp350 antigen expressed from Chinese hamster ovary (CHO) cells, and 16 of the antibodies reacted to both the 350 kDa and the 220 kDa splice variant. In contrast, HB2, HB3, HB6, HB7, HB13, HB20, and HB21 failed to recognize either of the denatured isoforms of gp350 ( FIG.  1 B ). The antibodies were further characterized by flow cytometric analysis of CHO cells stably expressing gp350 on the cell surface, and HB1, HB2, HB3, HB5, HB6, HB9, HB11, HB12, HB15, HB17, HB19, HB20, and HB21 antibodies readily recognized gp350 ( FIGS.  1 C,  1 D ). Given that HB2, HB3, HB20, and HB21 detected gp350 by flow cytometry, but not by immunoblot, suggests that these four antibodies recognized conformational epitopes on gp350, whereas HB5, HB9, HB11, HB15, HB17, and HB19 recognized both linear and conformational epitopes ( FIGS.  1 B- 1 D ). The observation that all 23 anti-gp350 antibodies recognized the gp350 antigen either by indirect ELISA, flow cytometry, or immunoblot assay suggests that antibodies that are specific to EBV gp350 protein were successfully produced. 
     Example 3: Analysis of the Variable Heavy and Variable Light Chain Sequences 
     The complementarity determining regions (CDRs) of the anti-gp350 mAbs were sequenced by amplifying mRNA representing unique heavy/light variable chain sequences using RT-PCR. RT-PCR products were sequenced and analyzed. The sequences of the heavy and light chain variable region genes (VH and VL, respectively) of the 23 new anti-gp350 antibodies, as well as the nAb-72A1, were determined and compared the sequences to published nAb-72A1 sequences (50, 51). The sequence of the CDR of this antibody was recently determined and published, revealing two unique IgG1 heavy chains and two unique light chains, one kappa and one lambda (50, 51). PCR was used to amplify the genes encoding the VH and VL chain regions in cDNA generated from the 23 hybridoma cells as well as from HB168 (nAb-72A1). The PCR products presented distinct bands at approximately 350-400 bp and 450-500 bp for VH and VL, respectively (data not shown). The purified fragments were sequenced using Illumina MiSeq, followed by in silico analysis and CDRs for both VH and VL were identified ( FIGS.  2 A,  2 B ). Two VH and VL sequences of nAb-72A1 were identified as &gt;94% identical to the previously published sequences (50), suggesting that nAb-72A1 exists as a mixed antibody, instead of the reported mAb (51). Similar to nAb-72A1, HB4, HB13, HB15, and HB23 hybridomas each produced a mixture of two antibodies, with unique sequences of the VH chain showing at &gt;5% frequencies, suggesting that they are not mAbs (Table 3). Coding sequences for VL chains for HB7, HB9, and HB17 were unable to be identified, unless the frequencies were lowered to &gt;1% (Table 3); in this case, the identified coding VL chain sequences were identical. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Summary of Illumina Dual Demultiplex of V H  and V L  Regions 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                 &gt;5% 
                   
               
               
                   
                   
                   
                 PEAR 
                 Length 
                 Primer 
                   
                 &gt;5% 
                 Unique 
               
               
                   
                   
                 Starting 
                 Merged 
                 Filtered 
                 Matched 
                 3x 
                 Unique 
                 Non- 
               
               
                 Sample 
                 Chain 
                 Pairs 
                 Reads 
                 Reads 
                 Reads 
                 Reads 
                 Coding 
                 Coding 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 HB1 
                 HEAVY 
                 51,641 
                 51,210 
                 46,655 
                 32,482 
                 22,725 
                 1 
                 0 
                   
               
               
                   
                 LIGHT 
                 280,048 
                 279,012 
                 100,725 
                 68,041 
                 58,570 
                 1 
                 1 
               
               
                 HB2 
                 HEAVY 
                 22,793 
                 22,621 
                 16,475 
                 11,415 
                 7,429 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 167,230 
                 166,496 
                 161,764 
                 132,752 
                 115,886 
                 1 
                 1 
               
               
                 HB3 
                 HEAVY 
                 26,382 
                 26,162 
                 25,542 
                 16,910 
                 11,709 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 12,681 
                 12,609 
                 11,753 
                 9,809 
                 8,023 
                 1 
                 1 
               
               
                 HB4 
                 HEAVY 
                 38,811 
                 38,238 
                 17,151 
                 11,957 
                 7,217 
                 2 
                 0 
                 § 
               
               
                   
                 LIGHT 
                 179,249 
                 129,752 
                 111,996 
                 78,392 
                 66,419 
                 1 
                 1 
               
               
                 HB.5 
                 HEAVY 
                 42,951 
                 42,173 
                 35,793 
                 25,842 
                 17,173 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 176,073 
                 175,267 
                 168,806 
                 141,712 
                 127,045 
                 1 
                 1 
               
               
                 HB6 
                 HEAVY 
                 26,142 
                 25,981 
                 22,245 
                 15,658 
                 10,453 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 171,996 
                 171,370 
                 167,730 
                 138,348 
                 122,397 
                 1 
                 1 
               
               
                 HB7 
                 HEAVY 
                 32,443 
                 32,094 
                 25,449 
                 17,615 
                 11,836 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 67,031 
                 63,924 
                 37,344 
                 26,271 
                 22,378 
                 1 
                 1 
                 * 
               
               
                 HB8 
                 HEAVY 
                 140,091 
                 103,349 
                 92,744 
                 58,292 
                 31,583 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 151,244 
                 115,527 
                 102,439 
                 82,154 
                 70,803 
                 1 
                 1 
               
               
                 HB9 
                 HEAVY 
                 37,057 
                 36,473 
                 19,544 
                 11,585 
                 7,358 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 409,432 
                 310,529 
                 136,074 
                 106,820 
                 90,063 
                 1 
                 2 
                 * 
               
               
                 HB10 
                 HEAVY 
                 38,181 
                 37,981 
                 26,043 
                 17,104 
                 11,391 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 114,255 
                 112,498 
                 106,914 
                 84,368 
                 75,370 
                 1 
                 1 
               
               
                 HB11 
                 HEAVY 
                 22,225 
                 21,841 
                 6,956 
                 4,408 
                 2,465 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 106,465 
                 102,278 
                 65,332 
                 50,232 
                 44,527 
                 1 
                 0 
               
               
                 HB12 
                 HEAVY 
                 83,044 
                 82,355 
                 46,350 
                 30,276 
                 20,886 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 53,098 
                 47,336 
                 15,823 
                 7,560 
                 5,845 
                 1 
                 1 
               
               
                 HB13 
                 HEAVY 
                 81,451 
                 80,372 
                 47,995 
                 32,216 
                 20,139 
                 2 
                 0 
                 § 
               
               
                   
                 LIGHT 
                 27,314 
                 24,774 
                 8,987 
                 5,457 
                 4,104 
                 2 
                 1 
               
               
                 HB14 
                 HEAVY 
                 76,299 
                 75,357 
                 28,309 
                 19,104 
                 12,939 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 153,011 
                 149,264 
                 48,474 
                 29,710 
                 25,133 
                 1 
                 1 
               
               
                 HB.15 
                 HEAVY 
                 26,551 
                 26,410 
                 16,387 
                 11,434 
                 7,002 
                 2 
                 0 
                 § 
               
               
                   
                 LIGHT 
                 78,525 
                 77,778 
                 43,509 
                 29,504 
                 24,731 
                 1 
                 1 
               
               
                 HB16 
                 HEAVY 
                 54,249 
                 53,943 
                 9,517 
                 7,128 
                 4,179 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 42,048 
                 40,351 
                 30,602 
                 22,758 
                 18,251 
                 2 
                 1 
                 § 
               
               
                 HB17 
                 HEAVY 
                 111,614 
                 110,882 
                 81,428 
                 50,844 
                 35,949 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 102,490 
                 100,488 
                 83,925 
                 65,925 
                 57,727 
                 1 
                 1 
                 * 
               
               
                 HB18 
                 HEAVY 
                 211,215 
                 155,410 
                 146,256 
                 91,009 
                 50,308 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 212,261 
                 161,879 
                 155,235 
                 123,096 
                 105,959 
                 1 
                 1 
               
               
                 HB19 
                 HEAVY 
                 109,692 
                 82,221 
                 20,546 
                 12,587 
                 7,274 
                 1 
                 1 
               
               
                   
                 LIGHT 
                 70,828 
                 69,744 
                 62,572 
                 48,354 
                 42,051 
                 1 
                 1 
               
               
                 HB20 
                 HEAVY 
                 15,781 
                 15,632 
                 12,789 
                 7,757 
                 4,852 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 135,527 
                 133,208 
                 118,513 
                 90,717 
                 78,701 
                 1 
                 1 
               
               
                 HB21 
                 HEAVY 
                 15,312 
                 15,202 
                 8,577 
                 5,645 
                 3,420 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 102,450 
                 100,171 
                 89,059 
                 68,552 
                 60,500 
                 1 
                 1 
               
               
                 HB22 
                 HEAVY 
                 217,959 
                 156,488 
                 154,008 
                 95,755 
                 50,245 
                 1 
                 0 
               
               
                   
                 LIGHT 
                 205,334 
                 156,986 
                 143,386 
                 108,728 
                 85,136 
                 1 
                 0 
               
               
                 HB23 
                 HEAVY 
                 196,390 
                 143,929 
                 123,028 
                 71,076 
                 39,358 
                 2 
                 0 
                 § 
               
               
                   
                 LIGHT 
                 158,594 
                 120,140 
                 115,476 
                 90,004 
                 78,787 
                 2 
                 0 
               
               
                 72A1 
                 HEAVY 
                 213,480 
                 158,199 
                 156,215 
                 107,395 
                 68,486 
                 2 
                 0 
                 § 
               
               
                   
                 LIGHT 
                 187,216 
                 140,964 
                 132,945 
                 105,783 
                 91,208 
                 1 
                 1 
               
               
                   
               
               
                 * Hybridoma with V L  chain sequences identified with &gt;1% frequency, § Hybridoma with more than one unique, plausible-coding V H  chain sequence with &gt;5% frequency. 
               
               
                 The term “unique” refers to unique sequence counts (so, identical sequences found in a substantial frequency of merged reads, not necessarily unique compared to other samples). 
               
            
           
         
       
     
     The analysis and comparison of the VH and VL chain gene sequences of the 23 hybridomas compared to HB168 (nAb-72A1) showed unique sequences within the CDR 1-3 region. Only HB8 and HB18 had identical VH and VL chain gene sequences, suggesting that the two are the same clone isolated separately; therefore, HB18 was excluded from subsequent experiments. One of the two HB15 antibodies had identical VH and VL gene sequences to that of HB10, however based on the previous characterization, the presence of the additional antibody in HB15 was sufficient to confer subtle differences in biochemical characterizations for gp350 between the two antibodies. Thus, based on the sequence analysis ( FIG.  2   ), 15 unique anti-gp350 mAbs were generated, with distinct biochemical and sequence identity from commercially available nAb-72A1. 
     Example 4: Neutralization Assay 
     The ability of the 15 mAbs (10 μg/ml or 50 μg/ml) to neutralize purified eGFP-tagged AGS-EBV infection of the Raji B cell line in vitro was evaluated following standardized procedures (52) and determined the percentage of eGFP+ cells using flow cytometry as described (45, 47). The nAbs 72A1 and E1D1 were used as positive controls, whereas the non-neutralizing mAb 2L10 was used as a negative control. Because HB4, HB7, HB13, HB15, HB16, HB19, HB21, and HB23 were confirmed to be mixtures based on isotyping or sequence data, they were eliminated from further consideration in the neutralization assay. An antibody was considered a neutralizer if it inhibited EBV infection &gt;20% at 10 μg/ml and &gt;60% at 50 μg/ml. Several mAbs inhibited EBV infection in a dose-dependent manner. HB20 and HB17 were the most effective at preventing EBV infection of Raji cells in vitro, whereby they reduced infection by 40% and &gt;60%, and &gt;30% and 80% at 10 μg/ml and 50 μg/ml, respectively ( FIGS.  3 A and  3 B ). The HB9 and HB10 antibodies prevented EBV infection of Raji cells by ˜25% at 10 μg/ml and ˜60% at 50 μg/ml. The HB11 antibody neutralized &lt;20% at 10 μg/ml, but showed a dose-dependent increase at 50 μg/ml by neutralizing EBV infection by 60%. By the set neutralization parameters, HB1-3, HB5-8, HB12, HB14, and HB22 did not neutralize EBV infection of Raji cells. In comparison, both nAb-72A1 and nAb-E1D1 neutralized EBV infection by &gt;70% and 40%, respectively, at 10 μg/ml. The nAb-72A1 neutralized EBV infection by &gt;95% at 50 μg/ml, whereas nAb-E1D1 neutralized infection by &gt;60% at 50 μg/ml. As expected, the negative control, mAb-2L10, did not neutralize viral infection at either concentration. Based on the neutralization assay results, the remaining 15 mAbs were organized into two distinct groups, neutralizers (+) and non-neutralizers (−) as summarized in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Summarized Biochemical and Functional Characterizations of Anti-gp350 Antibodies 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 ELISA 
                   
                   
                   
               
               
                   
                   
                   
                 binding to 
                 FACS 
               
               
                   
                 IgG 
                 Light 
                 purified EBV 
                 (CHO 
                 Western 
                 Neutralization 
               
               
                 Antibody 
                 sub-class 
                 chain 
                 gp350/220 
                 Cells) 
                 blot 
                 activity 
               
               
                   
               
               
                 HB1 
                 IgG1 
                 κ 
                 + 
                 + 
                 + 
                 − 
               
               
                 HB2 
                 IgG2a 
                 κ 
                 + 
                 + 
                 − 
                 − 
               
               
                 HB3 
                 IgG2a 
                 κ 
                 − 
                 + 
                 − 
                 − 
               
               
                 HB4 
                 IgG1 
                 κ 
                 + 
                 − 
                 + 
                 ND 
               
               
                 HB5 
                 IgG2a 
                 κ 
                 + 
                 + 
                 + 
                 − 
               
               
                 HB6 
                 IgG1 
                 κ 
                 − 
                 + 
                 − 
                 − 
               
               
                 HB7 
                 IgG1 
                 κ 
                 + 
                 − 
                 − 
                 − 
               
               
                 HB8 
                 IgG1 
                 κ 
                 + 
                 − 
                 + 
                 − 
               
               
                 HB9 
                 IgG2a 
                 κ 
                 − 
                 + 
                 + 
                 + 
               
               
                 HB10 
                 IgG1 
                 κ 
                 − 
                 − 
                 + 
                 + 
               
               
                 HB11 
                 IgG1 
                 κ 
                 + 
                 + 
                 + 
                 + 
               
               
                 HB12 
                 IgG1 
                 κ 
                 − 
                 + 
                 + 
                 − 
               
               
                 HB13 
                 IgG1 
                 κ 
                 + 
                 − 
                 − 
                 ND 
               
               
                 HB14 
                 IgG1 
                 κ 
                 + 
                 − 
                 + 
                 − 
               
               
                 HB15 
                 IgG1 
                 κ 
                 + 
                 + 
                 + 
                 ND 
               
               
                 HB16 
                 IgG/IgM 
                 κ 
                 + 
                 − 
                 + 
                 ND 
               
               
                 HB17 
                 IgG2b 
                 κ 
                 + 
                 + 
                 + 
                 + 
               
               
                 HB19 
                 IgG/IgM 
                 κ 
                 + 
                 + 
                 + 
                 ND 
               
               
                 HB20 
                 IgG2a 
                 κ 
                 − 
                 + 
                 − 
                 + 
               
               
                 HB21 
                 IgG1/IgG2b 
                 κ 
                 − 
                 + 
                 − 
                 ND 
               
               
                 HB22 
                 IgG1 
                 κ 
                 − 
                 − 
                 + 
                 − 
               
               
                 HB23 
                 IgG1 
                 κ 
                 − 
                 + 
                 + 
                 ND 
               
               
                 72A1 
                 IgG1 
                 κ 
                 + 
                 + 
                 + 
                 + 
               
               
                 E1D1 (anti gH/gL) 
                 IgG1 
                 κ 
                 − 
                 − 
                 − 
                 + 
               
               
                   
               
               
                 ND = Not determined, + = positive, − = negative, ELISA = enzyme-linked immunosorbent assay, κ = Kappa. 
               
            
           
         
       
     
     Example 5: Epitope Mapping 
     The 15 new anti-gp350 mAbs (neutralizers vs. non-neutralizers) were used to identify most, if not all, of the relevant immunodominant aa residues targeted by both the nAbs and non-nAbs. RepliTope approach was used, in which overlapping peptides [15-mer with 11 aa overlap] that cover the complete sequence of gp350 (aa 1-907) were immobilized on microarray slides and probed with the purified anti-gp350 antibodies in an ELISA format. nAb-72A1 was used as positive control, because the cognate epitopes bound by the antibody have previously been reported. 
     Two epitopes,  253 TPIPGTGYAYSLRLTPRPVSRFL 275  and  393 VSGLGTAPKTLIITRTATNATTT 415 , were both bound by all 15 mAbs, as well as nAb-72A1 and 2L10, regardless of their neutralizing or non-neutralizing capabilities. This consensus suggests that these epitopes are immunodominant. Several of the 15 mAbs (HB2, HB3, HB8, HB11, HB12, HB14, HB17, and HB22), as well as nAb-72A1, bound to  341 ANSPNVTVTAFWAWPNNTE 359 . Two epitopes, aa 341-359 and aa 393-415, were found within the previously identified single epitope II, which is encoded by nucleotides between 3,186 bp and 3,528 bp, corresponding to aa 326-439 of gp350 (56). Two mAbs, HB1 and HB10, bound  605 TTPTPNATGPTVGETSPQA 623 , an epitope located within the gp350 (501-699) splice region that is involved in generation of the 220 kDa splice variant. A total of eight mAbs (HB1-3, HB8, HB10-12, and HB22) also bound to the region between  1 MEAALLVCQYTIQSLIHLTGEDPG 24 , which includes a region homologous to C3d, another molecule known to interact with CD21 (51, 55). Two epitopes common between most nAbs and non-nAbs,  821 PPSTSSKLRPRWTFTSPPV 839  and  875 LLLLVMADCAFRRNLSTSHTYTTPPY 899 , were located upstream and downstream, respectively, of the transmembrane domain on the C-terminus of gp350. Epitope aa 821-839 is located within the previously identified epitope I, which is located between aa 733-841 (56). Furthermore, epitope aa 821-839 is potentially involved in EBV infection of B cells (58). However, the study could not identify two epitopes located at aa 282-301 and aa 194-211, which were previously shown to be involved in the binding of nAb-72A1 and CD21, respectively (51, 58, 59). It was shown that nAb-72A1 bound  145 EMQNPVYLIPETVPYIKWDN 164 , one of the neutralizing epitopes on gp350 (51, 58, 59). Nine gp350 epitopes identified by 15 neutralizing and non-neutralizing mAbs are shown in the sequence below and summarized in Table 5). Residues in bold represent the gp220 splice variant region. Residues in boxes represent RepliTope-identified epitopes and exact residues. Italic residues represent canonical neutralizing epitope, underlined residues represent epitope bound by all assayed mAb. 
     
       
         
           
               
               
               
            
               
                 1 
                 MEAALLVCQY TIQSLIHLTG EDPGFFNVEI PEFPFYPTCN VCTADVNVTI NFDVGGKKHQ 
                   
               
               
                   
               
               
                 61 
                 LDLDFGQLTP HTKAVYQPRG AFGGSENATN LFLLELLGAG ELALTMRSKK LPINVTTGEE 
               
               
                   
               
               
                 121 
                 QQVSLESVDV YFQDVFGTMW CHHA EMQNPV YLIPETVPYI KWD NCNSTNI TAVVRAQGLD 
               
               
                   
               
               
                 181 
                 VTLPLSLPTS AQDSNFSVKT EMLGNEIDIE CIMEDGEISQ VLPGDNKFNI TCSGYESHVP 
               
               
                   
               
               
                 241 
                 SGGILTSTSP VA TPIPGTGY AYSLRLTPRP VSRFL GNNSI LYVFYSGNGP KASGGDYCIQ 
               
               
                   
               
               
                 301 
                 SNIVFSDEIP ASQDMPTNTT DITYVGDNAT YSVPMVTSED ANSPNVTVTA FWAWPNNTET 
               
               
                   
               
               
                 361 
                 DFKCKWTLTS GTPSGCENIS GAFASNRTFD IT/ VSGLGTAP KTLIITRTAT NATTT THKVI 
               
               
                   
               
               
                 421 
                 FSKAPESTTT SPTLNTTGFA DPNTTTGLPS STHVPTNLTA PASTGPTVST ADVTSPTPAG 
               
               
                   
               
               
                 481 
                 TTSGASPVTP SPSPWDNGTE  SKAPDMTSST SPVTTPTPNA TSPTPAVTTP TPNATSPTPA   
               
               
                   
               
               
                 541 
                 
                   VTTPTPNATS PTLGKTSPTS AVTTPTPNAT SPTLGKTSPT SAVTTPTPNA TSPTLGKTSP 
                 
               
               
                   
               
               
                 601 
                 
                   TSAVTTPTPN ATGPTVGETS PQANATNHTL GGTSPTPVVT SQPKNATSAV TTGQHNITSS 
                 
               
               
                   
               
               
                 661 
                   STSSMSLRPS SNPETLSPST SDNSTSHMPL LTSAHPTGGE  NITQVTPASI STHHVSTSSP 
               
               
                   
               
               
                 721 
                 APRPGTTSQA SGPGNSSTST KPGEVNVTKG TPPQNATSPQ APSGQKTAVP TVTSTGGKAN 
               
               
                   
               
               
                 781 
                 STTGGKHTTG HGARTSTEPT TDYGGDSTTP RPRYNATTYL PPSTSSKLRP RWTFTSPPVT 
               
               
                   
               
               
                 841 
                 TAQATVPVPP TSPRRFSNLS MLVLQSASLA VLTLLLLLVM ADCAFRRNLS TSHTYTTPPY 
               
               
                   
               
               
                 901 
                 DDAETYV 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Antibody Binding gp350 Epitopes 
               
            
           
           
               
               
               
            
               
                   
                   
                 Epitope 
               
               
                 Epitope 
                 Amino acid sequence 
                 Properties 
               
               
                   
               
               
                 1 
                   1 MEAALLVCQYTIQSLIHLTGEDPG 24   
                   
               
               
                   
               
               
                 2 
                   145 EMQNPVYLIPETVPYIKWDN 164   
                 Neutralizing 
               
               
                   
               
               
                 3 
                   253 TPIPGTGYAYSLRLTPRPVSRF 
                 Immunodominant/ 
               
               
                   
                 L 275   
                 novel 
               
               
                   
               
               
                 4 
                   341 ANSPNVTVTAFWAWPNNTE 359   
                   
               
               
                   
               
               
                 5 
                   381 GAFASNRTFDIT 392   
                 Neutralizing/ 
               
               
                   
                   
                 novel 
               
               
                   
               
               
                 6 
                   393 VSGLGTAPKTLIITRTATNATT 
                 Immunodominant 
               
               
                   
                 T 415   
                   
               
               
                   
               
               
                 7 
                   605 TTPTPNATGPTVGETSPQA 623   
                   
               
               
                   
               
               
                 8 
                   821 PPSTSSKLRPRWTFTSPPV 839   
                   
               
               
                   
               
               
                 9 
                   875 LLLLVMADCAFRRNLSTSHTYTTP 
                 novel 
               
               
                   
                 PY 899   
               
               
                   
               
            
           
         
       
     
     Example 6: Construction of Chimeric Gp350 nAbs 
     Chimeric gp350 nAbs were constructed according to the diagrams of  FIG.  4   . First, mouse antibodies against human gp350 were developed, and then the mouse antibody variable region is fused to human constant region, for example, to human IgM. The heavy chain and light chain variable regions of the mouse antibody were cloned into pFUSE-CHIg and pFUSE2-CLIg vectors, respectively, followed by co-transfection of mammalian cells with recombinant pFUSE-CHIg and pFUSE2-CLIg vectors. The expression vectors were obtained from InvivoGen, and the expression was conducted in CHO cells or HEK293 cells, available from ATCC. The construction schemes and expression of heavy and light chains of clone 19 are shown in  FIG.  4   . 
     Analysis of VH-VL sequence from the HB168 (nAb-72A1) hybridoma revealed that the hybridoma produced two antibodies: one that is gp350-specific and another that recognizes mineral oil-induced plasmacytoma (MOPC) (57). To further investigate gp350 for additional neutralizing epitopes, the gp350-specific nAb-72A1 VH-VL sequence was used to generate chimeric (mouse/human) recombinant antibodies. Similarly, the VH-VL sequence for the HB20 antibody, which the neutralization analysis above showed to be one of the best nAb, was used to generate chimeric antibody. A negative control chimeric recombinant antibody was generated using VH-VL sequences from the gp350-specific but non-neutralizing HB5 antibody mentioned above ( FIG.  3   ). 
     Comparative analysis of the epitope binding pattern was performed using the chimeric recombinant antibodies and a novel epitope was revealed,  381 GAFASNRTFDIT 392 , which was bound by HB20 and nAb-72A1, but not by HB5, and is distinct from the  145 EMQNPVYLIPETVPYIKWDN 164  epitope, which is bound only by the nAb-72A1. The sequence below shows the novel gp350-neutralizing epitope identified by epitope mapping of neutralizing (nAb-72A1 and HB20) vs non-neutralizing (HB5) anti-gp350 mAbs. Epitopes bound by nAb-72A1 (double underlined), HB20 (wavy underlined), and HB5 (boxed) are indicated. Residues 501-700 represent splice variant region (bold), single underlined residues represent epitopes bound by nAb-72A1, HB20 and HB5, and italic residues represent epitopes bound by nAbs. 
     
       
         
           
               
               
               
            
               
                 1 
                   MEAALLVCQY TIQSL IHLTG EDPGFFNVEI PEFPFYPTCN VCTADVNVTI NFDVGGKKHQ 
                   
               
               
                   
               
               
                 61 
                 LDLDFGQLTP HTKAVYQPRG AFGGSENATN LFLLELLGAG ELALTMRSKK LPINVTTGEE 
               
               
                   
               
               
                 121 
                 QQVSLESVDV YFQDVFGTMW CHHA   EMONPV YLIPETVPYI KWDN   CNSTNI TAVVRAQGLD 
               
               
                   
               
               
                 181 
                 VTLPLSLPTS AQDSNFSVKT EMLGNEIDIE CIMEDGEISQ VLPGDNKFNI TCSGYESHVP 
               
               
                   
               
               
                 241 
                 SGGILTSTSP VA TPIPGTGY AYSLRLTPRP VSRFL GNNSI LYVFYSGNGP KASGGDYCIQ 
               
               
                   
               
               
                 301 
                 SNIVFSDEIP ASQDMPTNTT DITYVGDNAT YSVPMVTSED  ANSPNVTVTA FWAWPNNTET   
               
               
                   
               
               
                 361 
                   DFK CKWTLTS GTPSGCENIS    VSGLGTAP KTLIITRTAT NATTT THKVI 
               
               
                   
               
               
                 421 
                 FSKAPESTTT SPTLNTTGFA DPNTTTGLPS STHVPTNLTA PASTGPTVST ADVTSPTPAG 
               
               
                   
               
               
                 481 
                 TTSGASPVTP SPSPWDNGTE  SKAPDMTSST SPVTTPTPNA TSPTPAVTTP TPNATSPTPA   
               
               
                   
               
               
                 541 
                 
                   VTTPTPNATS PTLGKTSPTS AVTTPTPNAT SPTLGKTSPT SAVTTPTPNA TSPTLGKTSP 
                 
               
               
                   
               
               
                 601 
                 
                   TSAVTTPTPN ATGPTVGETS PQANATNHTL GGTSPTPVVT SQPKNATSAV TTGQHNITSS 
                 
               
               
                   
               
               
                 661 
                   STSSMSLRPS SNPETLSPST SDNSTSHMPL LTSAHPTGGE  NITQVTPASI STHHVSTSSP 
               
               
                   
               
               
                 721 
                 APRPGTTSQA SGPGNSSTST KPGEVNVTKG TPPQNATSPQ APSGQKTAVP TVTSTGGKAN 
               
               
                   
               
               
                 781 
                 STTGGKHTTG HGARTSTEPT TDYGGDSTTP RPRYNATTYL PPSTSSKLRP RWTFTSPPVT 
               
               
                   
               
               
                 841 
                 TAQATVPVPP TSQPRFSNLS MLVLQWASLA VLTLLLLLVM  ADCAFRRNLS TSHTY TTPPY 
               
               
                   
               
               
                 901 
                 DDAETYV 
               
            
           
         
       
     
     Example 7: Development of Antibody-Small Molecule Conjugates (ADCs) 
     Using small molecule L2P4 as an example, antibody-small molecule conjugates can be developed as illustrated in  FIG.  5   . One or more small molecules can be conjugated to the antibody heavy chain or light chain via a reactive donor group and a C-terminal acceptor group. Small molecule L2P4 was disclosed in the publication by Jiang et al. 40  After validating the function of the purified chimeric gp350 nAbs using ELISA, flow cytometry (FC), and surface plasmon resonance, the nAbs can be conjugated to L2P4 in a site-specific manner via a Val-Cit dipeptide linker, which releases the active agent upon internalization of the ADC. 41, 42    
     Example 8: Efficacy Test of ADCs 
     To screen for optimal dose and identify the best nAb clone for pre-clinical studies, the ability of the ADC to neutralize EBV in vitro and to protect against PTLD in vivo is tested using a humanized mouse, as described 43,44 . In brief, purified chimeric ADC (or controls: PBS, or isotype-matched non-nAbs) is injected by I.V. into humanized mice, followed by EBV-B95-8-eGFP challenge. Mice are monitored regularly and euthanized upon signs of illness or after a preset limit of 100 days. Routine histology and necropsy are conducted to assess the efficacy of the ADC to protect against EBV infection and PTLD development. 
     REFERENCES 
     The references listed below, and all references cited in the specification are hereby incorporated by reference in their entireties, as if fully set forth herein.
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