The Epstein–Barr virus (EBV) is the first reported oncogenic herpesvirus that establishes persistent infection in B lymphocytes in 95% of adults worldwide. Glycoprotein B (gB) plays a predominant role in the fusion of the viral envelope with the host cell membrane. Hence, it is of great significance to isolate gB-specific fusion-inhibiting neutralizing antibodies (NAbs). AMMO5 is the only gB NAb but fails to antagonize B-cell infection. It is essential to isolate potent NAbs that can completely block EBV infection of B cells. Using hybridoma technology and neutralization assay, we isolate two gB NAbs 8A9 and 8C12 that are capable of completely neutralizing B-cell infection in vitro. In addition, 8A9 shows cross-reactivity with rhesus lymphocryptovirus (rhLCV) gB. Competitive binding experiments demonstrate that 8A9 and 8C12 recognize novel epitopes that are different from the AMMO5 epitope. The epitopes of 8A9 and 8C12 are mapped to gB D-II, and the AMMO5 epitope is located precisely at gB aa 410–419. We find that 8A9 and 8C12 significantly inhibit gB-derived membrane fusion using a virus-free fusion assay. In summary, this study identifies two gB-specific NAbs that potently block EBV infection of B cells. Our work highlights the importance of gB D-II as a predominant neutralizing epitope, and aids in the rational design of therapeutics or vaccines based on gB.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that establishes lifelong persistence and is related to multiple diseases, including cancers. Neutralizing antibodies (NAbs) have proven to be highly effective in preventing EBV infection and subsequent diseases.

Epstein–Barr virus (EBV) is the first reported oncogenic virus and infects more than 90% of adults worldwide. EBV can establish a latent infection in B lymphocytes which is essential for persistence and transmission. Glycoprotein gp42 is an indispensable member of the triggering complex for EBV entry into a B cell. The N-terminal region of gp42 plays a key role in binding to gH/gL and triggering subsequent membrane fusion. However, no antibody has been reported to recognize this region and the immunogenicity of gp42 N-domain remains unknown. In the present study, we have generated a panel of nine mAbs against the gp42 N-terminal region (six mAbs to gp42-44-61aa and three mAbs to gp42-67-81aa). These mAbs show excellent binding activity and recognize different key residues locating on the gp42 N-domain. Among the nine mAbs, 4H7, 4H8 and 11G10 cross-react with rhLCV-gp42 while other mAbs specifically recognize EBV-gp42. Our newly obtained mAbs provide a useful tool for investigating the gp42 function and viral infection mechanism of γ-Herpesvirus. Furthermore, we assess the immunogenicity of the gp42 N-terminal region using the HBc149 particle as a carrier protein. The chimeric VLPs can induce high antibody titers and elicit neutralizing humoral responses to block EBV infection. More rational and effective designs are required to promote the gp42-N terminal region to become an epitope-based vaccine.

Humanized mouse models are used as comprehensive small-animal models of EBV infection. Previously, infectious doses of EBV used in vivo have been determined mainly on the basis of TD₅₀ (50% transforming dose), which is a time-consuming process. Here, we determined infectious doses of Akata-EBV-GFP using green Raji units (GRUs), and characterized dose-dependent effects in humanized mice. We defined two outcomes in vivo, including an infection model and a lymphoma model, following inoculation with low or high doses of Akata-EBV-GFP, respectively. Inoculation with a low dose induced primary B cells to become lymphoblastoid cell lines in vitro, and caused latent infection in humanized mice. In contrast, a high dose of Akata-EBV-GFP resulted in primary B cells death in vitro, and fatal B cell lymphomas in vivo. Following infection with high doses, the frequency of CD19⁺ B cells decreased, whereas the percentage of CD8⁺ T cells increased in peripheral blood and the spleen. At such doses, a small part of activated CD8⁺ T cells was EBV-specific CD8⁺ T cells. Thus, GRUs quantitation of Akata-EBV-GFP is an effective way to quantify infectious doses to study pathologies, immune response, and to assess (in vivo) the neutralizing activity of antibodies raised by immunization against EBV.