Neutralizing Antibodies to Epstein Barr Virus in the Rhesus Macaque Animal Model and in Humans

Abstract

Epstein-Barr virus (EBV) is associated with a number of human diseases and does not have a vaccine. It is believed that neutralizing antibodies are an important immune effector for an EBV vaccine, but it is unknown whether serum neutralizing antibodies can alter EBV infection through the oral mucosa. The studies presented in this dissertation were designed 1) to adapt the rhesus macaque animal model to allow testing of neutralizing antibodies in a biologically relevant system and 2) to better define the neutralizing antibody response of EBV infected humans. Infection of rhesus macaques with the EBV related lymphocryptovirus, rhLCV, provides an accurate model system for studying EBV, but there were two hurdles that needed to be overcome before neutralizing antibodies could be tested in this model. First, there are no neutralizing antibodies specific to rhLCV and we found that a potent EBV neutralizing antibody, 72A1, did not cross-neutralize rhLCV. Second, murine monoclonal antibodies are inherently immunogenic in macaques and induce anti-antibody responses that limit their utility. To create a virus sensitive to 72A1 neutralization, the major membrane glycoprotein of rhLCV with replaced with EBV gp350. The data presented here show that this chimeric virus can use EBV gp350 to support entry into macaque cells in vitro and following oral inoculation of rhesus macaques. To reduce the immunogenicity of the murine antibodies, “rhesusized” antibody variants were generated and shown to retain antigen specificity. The combination of this novel, chimeric virus and the “rhesusized” antibodies will now allow testing of neutralizing antibodies in the macaque model. Although multiple EBV glycoproteins have been shown to induce neutralizing antibodies in mice, studies of the human neutralizing antibody response have been narrowly focused on a single antibody binding epitope on gp350. Here we show that antibodies binding to this epitope do not represent all EBV neutralizing activity in human sera. Additionally, these data suggest that the neutralizing response is much broader than appreciated, with multiple glycoproteins inducing EBV neutralizing antibodies. Accurately defining the repertoire of viral glycoproteins targeted by human neutralizing antibodies can inform us of the naturally immunogenic proteins that may make good vaccine immunogens.