THE ROLE OF EXOSOMES IN MAREK`S DISEASE VIRUS PATHOGENESIS AND IMMUNITY

Marek's disease (MD) is an immunosuppressive and oncogenic pathology of chickens caused by Marek's disease virus (MDV). Losses due to MD are controlled through the near ubiquitous use of vaccines composed of chicken fibroblasts infected with non-oncogenic related viruses or live attenuated viruses. Although in much of poultry production, chickens are vaccinated prior to hatch, at 18 days of embryonation, the vaccines confer lifelong protection to tumor formation caused by field strains of MDV, which are present in many, if not all chicken houses. Vaccine viruses replicate for a relatively short time in the host (days to a few weeks) yet confer protection via eliciting systemic cell-mediated immunity. In our first round of funding, we found that exosomes (small double-membrane vesicles produced by nearly all cells) in the serum of vaccinated and protected chickens (VEX) contained mRNAs encoding most of the open reading frames of MDV field strains, despite no sites of vaccine virus replication being identified by this stage of infection (7 weeks post-hatch). In the serum of tumor-bearing chickens, we found that these tumor-associated exosomes (TEX) contained mRNAs primarily for the oncogene-encoding regions of the virus. This presented the attractive hypothesis that systemic lifelong protection conferred by MD vaccines does so by having VEX fuse with antigen-presenting cells to have individual structural proteins translated, processed for MHC-I presentation and activation of cytotoxic T-cells in the absence of vaccine virus replication. To follow up on this hypothesis, we have examined what cells are likely to take up these VEX and TEX (monocytes, macrophages, dendritic cells) using the HD11 cell line patterned to be each of these cell lineages. We have performed proteomic analysis of these cells: (1) as they are patterned to become these lineages, (2) after treatment with VEX and TEX, and (3) what changes in metabolism and cell proliferation occur during this patterning. Additionally, we examined the effect of VEX and TEX treatment on vaccine efficacy in anin vivochallenge model, and examined the timing of vaccine virus mRNA accumulation in VEX using SPF chickens in an isolator study.In renewal of this funding, we seek to: (1) Determine if peptides from VEX-encoded mRNAs are presented on the surface of antigen presenting cells, (2) Determine if VEX-treated antigen presenting cells can elicit proliferation of CTLs from syngeneic vaccinated chickens, (3) Optimize the use of VEX to increase MD vaccine efficacy, and (4) Determine the virus-host interactions leading to the release of MDV mRNAs from infected host cells (macrophages, dendritic cells, etc.). Completion of this work will provide insight into a fundamental mechanism of lifelong, systemic anti-viral/anti-tumor immunity that may have far-reaching applications in animal, and perhaps human health.