EBNA1-specific T cell responses during persistent rhesus LCV infection and the development of a novel therapeutic prototype vaccine for EBV-associated diseases

The impact of EBV on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA1) is an important target for vaccination because it is the only protein expressed in all forms of latency and in all EBV-associated malignancies. The overarching goal throughout this dissertation was to determine if EBNA1 is a suitable target for vaccine development. This was addressed in two ways. First, because an improved understanding of EBNA1-specific T cell responses benefits EBV vaccine development, we characterized responses against EBNA1 of the EBV-homologous rhesus lymphocryptovirus (rhLCV) in naturally infected rhesus macaques. We assessed frequency, phenotype, and cytokine production profiles of rhesus (rh)EBNA1-specific T cells by intracellular cytokine staining (ICS) and polychromatic flow cytometry. We found that most naturally infected animals had CD4+ and/or CD8+ T cells against rhEBNA1 and rhBZLF1, an immediate-early lytic phase antigen of rhLCV. Peptide-specific CD8+ T cells showed a more activated effector phenotype, while most peptide-specific CD4+ T cells exhibited a resting central memory phenotype. T cells were highly functional and produced various combinations of the cytokines IFN-γ, IL-2, and TNF-α. The differentiation status and functional profiles of rhEBNA1-specific T cells suggests they are not impaired by chronic exposure to low levels of antigen, and rhEBNA1-specific T cells therefore represent a viable population to target through vaccination. Similarities between our findings and the human response further validate the rhLCV model for studying chronic EBV infection and for pre-clinical vaccine development. We then asked if vaccination could expand functional rhEBNA1-specific T cells during persistent rhLCV infection. To test this, we developed two serologically distinct replication-defective adenoviral vectors that expressed chimeric rhEBNA1 constructs fused to functional and non-functional versions of Herpes Simplex Virusglycoprotein D (HSV-gD). HSV-gD has been shown to augment T cell responses by inhibiting the immunosuppressive Herpes Virus Entry Mediator (HVEM) pathway during T cell activation. After confirmation of vaccine specificity and antigenicity in vitro, rhesus macaques were vaccinated in a prime-boost regimen, and responses in peripheral blood were measured by ICS and polychromatic flow cytometry. Importantly, we found that vaccination induced the expansion of highly functional rhEBNA1-specific CD8+ and CD4+ T cells in vivo, regardless of HSV-gD binding ability. Vaccination did not increase rhBZLF1-specific T cell responses, thus indicating that rhEBNA1-specific responses were vaccine-driven. Overall, these results serve as important proof-of-principle analyses of a therapeutic EBNA1-based vaccine regimen and demonstrate that EBNA1 is a viable target that warrants exploration in future vaccine studies. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) This dissertation is available at ScholarlyCommons: http://repository.upenn. edu/edissertations/1343 Graduate Group Cell & Molecular Biology First Advisor Hildegund C. Ertl