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Identification of a Novel Neutralizing and Two Non-Neutralizing Epitopes on Epstein-Barr Virus gp350 Protein

Prevention of Epstein-Barr virus (EBV) primary infection has focused on generating neutralizing antibodies (nAbs) targeting the major envelope glycoprotein gp350/220 (gp350). To date, eight gp350 epitopes have been identified, but only one has elicited nAbs. In this study, we generated 23 hybridomas that produced anti-gp350 antibodies. We compared the candidate anti-gp350 antibodies to nAb-72A1 by: (1) testing their ability to detect gp350 using ELISA, flow cytometry, and immunoblot; (2) sequencing their heavy and light chain complementarity-determining regions (CDRs); (3) measuring the ability of each monoclonal antibody (mAb) to neutralize EBV infection in vitro; and (4) mapping the gp350 amino acids bound by the mAbs using RepliTope peptide microarrays. Eight antibodies recognized both denatured and non-denatured gp350, whereas five failed to react with denatured gp350 but recognized native gp350, suggesting they recognized conformational epitope(s). Sequence analysis of the heavy and light chain variable regions of the hybridomas identified 15 as mAbs with novel CDR regions unique from those of nAb-72A1. Seven of the new mAbs neutralized EBV in vitro, with HB20 and HB17 reducing EBV infection by 40% and >60%, and >30% and 80%, at 10 μg/ml and 50 μg/ml, respectively. Epitope mapping identified nine epitopes and defined their core residues, including two unique immunodominant epitopes, 253TPIPGTGYAYSLRLTPRPVSRFL253 and 875LLLLVMADCAFRRNLSTSHTYTTPPY899, and a novel nAb epitope 381GAFASNRTFDIT392. This study provides comprehensive in vitro mapping of the exact residues defining nine epitopes of EBV gp350. Our findings will inform novel strategies to design optimal EBV vaccines capable of conferring broader protection against the virus. Importance Neutralizing antibodies (nAbs) directed against Epstein-Barr virus envelope glycoprotein gp350/220 (gp350) are generated in humans upon infection or immunization, and are thought to prevent neonatal infection. However, clinical use of exogenous nAbs (passive immunization) is limited to a single study using the only well-characterized nAb, 72A1. The gp350 ectodomain contains at least eight unique B-cell binding epitopes; two of these epitopes are recognized by nAb-72A1. The exact amino acid residues of the other six epitopes and their role in generating nAbs has not been elucidated. We used our 15 newly generated and fully characterized monoclonal antibodies and a peptide-overlapping RepliTope array to provide a comprehensive map of the core amino acid residues that define epitopes of gp350 and to understand their role in generating nAbs. These results will inform design of better-targeted gp350 peptide vaccines that contain only protective epitopes, which will focus the B-cell response to produce predominantly nAbs.