Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717
Performing Department
(N/A)
Non Technical Summary
Marek's disease (MD) is a cancer of chickens for which a vaccine injected into the eggs of chickens prior to hatch provides lifelong prevention of this cancer. MD is caused by a virus (Marek's disease virus, MDV) and the vaccines that prevent MD are composed of related viruses that do not cause disease. How these related viruses train the immune system to provide such a longlasting anti-viral/anti-cancer response is currently unknown, as the vaccine viruses have very limited replication in commercial chickens due to maternal antibodies and their having been bred for resistance to MD. It is our hypothesis that small vesicles (the size of viruses) produced by some infected cells in the body of the chickens provide this lifelong protection from MD. These small vesicles, termed exosomes, contain proteins, lipids, DNAs and RNAs that we believe carry bits of the virus which then provide these to specialized cells of the immune system that pattern the way the body responds to challenge in the field.As disease-causing MDVs can be found in most, if not all chicken houses in the US, in the form of infectious dander (dead skin cells), challenge with MDV is considered to be highly likely during the life of the chicken.Exosomes are incredibly prevalent (~ten billion per ml of serum) in all biological fluids (serum, tears, urine, semen, lymph, etc.). To understand how exosomes confer immune protection, we have been examining their uptake by a phagocytic cell line that has been treated with chemicals and proteins to become dendritic cells (DCs), a population essential for the proper patterning of immunty. In the proposed work, we try to identify whether these cells, once treated with exosomes from the serum of birds which have been vaccinated and have survived challenge with highly-virulent MDVs, will stimulate immune cells to become activated. We also plan to determine if these exosomes can work together with vaccines or can be used as vaccines themselves to augment or prevent disease in chickens that will then be challenged with a highly-virulent MDV.In addition, since cancer cells produce exosomes that have been associated with progression and dissemination (metastasis), we plan to use exosomes isolated from the serum of tumor-bearing chickens to determine if these alter or prevent appropriate patterning of the immune response. To do this, we will be treating the phagocytic cell line (in the various forms patterned by cytokines) with known agonists (specific inducers) of different types of innate immune responses (inflammation, interferon responses, etc.) and then measure their effects compared to non-treated controls (for which we have significant data showing what a normal response would be). In addition, we plan to determine if administration of these exosomes from tumor-bearing birds affects MD vaccines and prevents them from providing protection from challenge.Finally, we want to try and understand what cells in the body are infected by MDV but then produce the exosomes which help to provide protection. Preliminary studies suggest that phagocytic cells do in fact become infected, but that they do not become infectious (able to transmit infection). We plan to study this virus-host cell interaction in order to better understand how MD vaccines produce exosomes that give rise to systemic protection. This work is fundamental to our understanding of how MD vaccines work and my yield insight into the development of vaccines for human virus-induced malignancies.
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Goals / Objectives
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.
Project Methods
From our overall specific aims, we plan the following produts and outputs:Aim 1.Determine if peptides from VEX-encoded mRNAs are presented on the surface of antigen presenting cells:This aim will yield publications on the proteomes of patterned HD11 cells, proteomes of vaccine-associatedexosome-treated HD11 cells, and surface MHC-I proteomes of HD11 cells and monocytes/macrophages collected from vaccinated and protected chickens.Aim 2.Determine if VEX-treated antigen presenting cells can elicit proliferation of CTLs from syngeneic vaccinated chickens:This aim will yield data and publications on the functional role of patterned HD11 cells and their ability to stimulate proliferation of MHC-I matched CD8+ T-cells from vaccinated chickens and identify T-cell epitopes presented in the context of MHC-I and MHC-II conferred by treatment with exosoes..Aim 3.Optimize the use of VEX to increase MD vaccine efficacy: In this aim, we will determine the optimal time and dose of VEX injection to increase vaccine efficacy using a sub-optimal vaccine (HVT only) in combination with a very virulent MDV challenge (RB-1B). By using a matrix of Vex treatment doses and administration times, we will determine the empirically the best time for stimulation of cell-mediated responses to MDV infection. Aim4. Determine the effect of TEX on impairing both innate and acquired immune responses. For this aim, patterned HD11 cells will be pre-treated with TEX (and VEX) and then treated with innate immune agonists that induce inflammatory cytokine gene expression, interferon and interferon inducible genes and one that induce both. Responses will be measured by qRT-PCR and compared to vehicle-only treated samples. To translate the observed effects to in vivo studies, we will conduct vaccine response trials similar to those described in Aim 3. In fact, these will be done in concert with the studies in Aim 3 to have directly comparable challenge virus administration. In this aim, a highly protective vaccine will be administered followed by TEX at an array of doses and timing followed by challenge with a virus strain for which the vaccineshould protect the chicken from developing disease.Additional Aim: During our studies, we plan to determine what cell types are harboring MDV in vivo (with cell culture models, as well) and generating the exosomes containing MDV-encoded mRNAs. These data will provide information regarding how the virus-cell interaction leads to a non-classically productive infection of this cell type, but yields these exosomes. We have preliminary data that suggest that macrophages have this capacity.