Marek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more virulent MDV strains have repeatedly emerged in vaccinated flocks. Thus, there is a need to understand how MD vaccines work in order to design future vaccines that are more protective, especially against more virulent MDVs. One promising insight for vaccine development is based upon protective synergism, a phenomenon where two vaccines when combined provide greater protection compared to either original vaccine when administered alone as a monovalent vaccine. The mechanism that underlines the synergistic effect between SB-1 (a Gallid herpesvirus 3, aka, serotype 2 strain) and HVT (herpesvirus of turkey, aka, Meleagrid herpesvirus 1 or serotype 3), two of the most widely used MD vaccines, has never been investigated, and thus, provides a highly relevant and useful model to explore. To investigate the mechanisms of protective synergy of SB-1 and HVT, we used three approaches. First, we investigated how monovalent SB-1 or HVT replicates when they were alone in the host or together as a bivalent vaccine. We observed that the replication patterns of SB-1 and HVT were different with respect to time after administration into the bird and the organs that they were found to replicate in regardless if the other vaccine were present. Based on the observation that HVT replicated primarily early in the bursa, we found that this organ was necessary for protection using both HVT and bivalent HVT + SB-1 vaccines. Second, we measured the effects of CD8 T cells in monovalent SB-1, HVT, and bivalent SB-1+HVT vaccine treatment. Specifically, we reduced CD8 T cells to see their effect of CD8 T cells on MD incidence and vaccinal protection by injecting the chickens with a monoclonal antibody directed against chicken CD8 T cells. In this study, we found that CD8 T cells were necessary for protection induced by vaccines. Third, we identified the cytokine profiles induced by SB-1, HVT, and the bivalent vaccine to see if cytokine synergy could be one of the mechanisms to explain protective synergy. We found that SB-1 induced an innate anti-viral response typified by IFN-α, IFN-β, IL-1β, T-cell proliferation cytokine IL-21, and Th2 cytokine IL-5, while HVT suppressed TGF-β3 and TGF-β4. The early stimulation of IL-1β and IL-21 (IFN-γ-promoting cytokines) at 4 days post vaccination (DPV) by SB-1 combined with the suppression of TGF-β (IFN-γ- suppressing cytokine) at 1 day post challenge (DPC) by HVT could result in the strong induction of IFN-γ found in the bivalent vaccine at 10 DPC. The induction of IFN-γ supports the synergistic effect of cytokines by a cooperative action mechanism where multiple cytokines work together to enhance the signal. Based on these findings, we propose a model to explain bivalent SB-1 and HVT vaccine synergy, which combines the replication of vaccines, T cell response to vaccinations, and cytokine synergy between SB-1 and HVT vaccine. Our proposed mechanism provides insights on how to generate rationally designed MD vaccines.
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