Double trouble: epithelial and B cell neutralization of Epstein-Barr virus

McGuire Laboratory, Vaccine and Infectious Disease Division

The Epstein-Barr virus (EBV), of the herpes family, is one of the most common human viruses. Most adults have been infected over the course of their life. Infections are mostly asymptomatic, however EBV can lead to infectious mononucleosis. Once a person is infected, the virus becomes inactive and enters latency, which can become a problem if reactivation occurs in immunocompromised populations. EBV is interesting in that it targets two different cell types by different means. Both B cells and epithelial cells are targeted in primary infection, binding of the virus and fusion with the membrane is mediated by three viral glycoproteins, gH, gL, and gB. In brief, the heterodimeric gH/gL complex interacts with the viral receptor which then triggers gB mediated fusion and thus infection. On epithelial cells the receptors for EBV have been identified as the ephrin receptor A2 (EphA2) and intergrins avb5, avb6, and avb8. In order for EBV to infect B cells another viral glycoprotein is needed. Entry is triggered by gH/gL complexing with gp42 which interacts with the human leukocyte antigen (HLA) class II on B cells leading to fusion by gB. In addition to these glycoproteins EBV also has gp350, which is the most abundant surface protein and aids in attachment through interactions with CD21 on target cells. Even though researchers understand how EBV infects cells, there are no vaccines to prevent infection. In addition, previously described antibodies are murine and either have not been well characterized or only neutralize entry into one of the two cell types. This gap in knowledge led researchers from the Vaccine and Infectious Disease Division at Fred Hutch to initiate a study to isolate and characterize human antibodies against EBV. Dr. McGuire and his team recently published these findings in Immunity.

Schematic of methods used to identify AMMO1 antibody. Human EBV-specific antibodies were isolated using EBV glycoproteins as bait. AMMO1 was identified, produced and found to neutralize infection in both primary cell types by binding to the junction between gH/gL. Image provided by Dr. McGuire.

By using individual glycoproteins as baits, the group was able to find EBV specific antibodies in the blood of three donors. Antigen positive B cells were sorted onto feeder cells, cultured and supernatants were then tested using an ELISA. From these sorts the cells yielded two- gH/gL specific and seven- gB specific B cells that were then sequenced for heavy and light chain transcripts, leading to pairs for one gH/gL (AMMO1) and four gB (AMMO2-5) antibodies. These paired antibodies were then cloned into recombinant expression vectors, expressed, and tested to confirm binding. After binding confirmation, the antibodies were tested in a neutralization assay in both B cells and epithelial cells resulting in AMMO1 being the only antibody to neutralize both cell types and AMMO5 neutralizing only epithelial cells (AMMO2-4 did not neutralize either cell type). Compared to other previously published gH/gL antibodies, AMMO1 showed 1-2 orders of magnitude more binding. Using cryoEM and competition assays the group found that AMMO1 binds a unique epitope spanning residues on both gH and gL. This residue partially overlaps with CL40 (a published antibody) binding epitope.  CL40 inhibits gp42 binding by sterically displacing it from the gH/gL complex. To test this for AMMO1 the group looked at binding of this complex to the cell surface and if that mediated fusion. In both cell types, preincubation with AMMO1 reduced binding of the glycoprotein complex to the cell surface but did not completely inhibit it. Fusion was accessed following binding, and the authors found that incubation with AMMO1 exhibited significantly reduced cell fusion. This data suggests that AMMO1 restricts viral access through gH/gL to the cell surface and blocks fusion, thus leading to viral neutralization. This dual-tropic neutralization of EBV primary target cells marks AMMO1 as a possible therapeutic agent, potentially using the antibody for passive transfer in high-risk groups. These results also support gH/gL subunit vaccines as a possible target for antibody production that can lead to dual neutralizing antibodies. In conclusion, Dr. McGuire stated, “We hope to use this information to design a safe and effective EBV vaccine. Moreover, this is the first human antibody that neutralizes EBV infection, subsequently we are exploring whether it might have any therapeutic use against EBV-related complications in immunocompromised individuals, like transplant recipients.”

Snijder J, Ortego MS, Weidle C, Stuart AB, Gray MD, McElrath MJ, Pancera M, Veesler D, McGuire AT. 2018, An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus. Immunity. 48(4):799-811.e9. doi: 10.1016/j.immuni.2018.03.026.

Funding provided by the Fred Hutch, the National Institutes of Health, Pew Biomedical Scholars Award, Netherlands Organization for Scientific Research and the European Molecular Virology Organization.