Mechanisms of "immune paralysis" caused by filoviruses

感染丝状病毒埃博拉病毒(EBOV)和马尔堡病毒(Marv)，导致以T细胞反应不足和淋巴细胞减少为特征的“免疫瘫痪”，尽管没有T细胞的感染，但也会导致严重的出血热，死亡率高达80%。在过去的十年里，研究人员发现了丝状病毒蛋白介导的I型干扰素(IFN-I)拮抗的多种机制。然而，丝状病毒迅速和有害地使适应性免疫反应失效的机制仍不清楚。 树突状细胞(DC)是专职的抗原提呈细胞，被认为很容易被感染，但不会经历正常的成熟。我们的初步数据表明，EBOV引起的干扰素-I拮抗作用与DC成熟不足之间存在很强的联系。该方案的中心假设是，丝状病毒蛋白和多种机制介导的干扰素-I反应的异常强烈和多余的拮抗作用阻碍了DC的成熟，导致T细胞的缺陷和/或异常刺激。这一假说将通过追求三个特定目标来验证：1)确定干扰素拮抗域和干扰素-I信号在DC成熟和T细胞激活、增殖和凋亡中的作用；2)确定感染DC分泌的可溶性因子和特定的抑制和促凋亡分子在T细胞激活、增殖和凋亡中的作用；3)确定丝状病毒感染时抑制T细胞激活和增殖或T细胞凋亡的作用是否可以通过诱导DC成熟来逆转。该项目将涉及用重组EBOV感染来自捐献者血液的DC，其突变体的干扰素-I拮抗域被禁用，或MARV；刺激原代人类CD4+和CD8+T细胞；通过深度测序表征丝状病毒感染和干扰素拮抗结构域对受感染DC和受刺激T细胞转录的影响；阻断干扰素-I信号和免疫抑制和促凋亡途径；以及试图用细胞因子鸡尾酒挽救受感染DC的成熟。总之，这些实验代表了一种广泛的、综合的方法来研究丝状病毒使适应性免疫反应失效的机制。

Infections with filoviruses Ebola (EBOV) and Marburg (MARV), lead to the "immune paralysis" characterized by a deficient T cell response and lymphopenia, despite the lack of infection of T cells, and a severe hemorrhagic fever with up to 80% mortality. Over the past decade, researchers identified multiple mechanisms of type I interferon (IFN-I) antagonism mediated by filovirus proteins. However, the mechanisms of the rapid and deleterious disabling of the adaptive immune response by filoviruses remain unknown. Dendritic cells (DC), which are the professional antigen-presenting cells, are believed to be readily infected, but do not undergo normal maturation. Our preliminary data have demonstrated the strong connection between IFN-I antagonism caused by EBOV and the lack of DC maturation. The central hypothesis of the proposal is that the unusually strong and redundant antagonism of the IFN-I response mediated by filovirus proteins and by multiple mechanisms, blocks maturation of DC, resulting in the deficient and/or aberrant stimulation of T cells. The hypothesis will be tested by pursuing three specific aims: 1) Determine the role of IFN-antagonizing domains and IFN-I signaling in the maturation of DC and in the activation, proliferation and apoptosis of T cells; 2) Determine the role of soluble factors secreted by the infected DC and specific inhibitory and pro-apoptotic molecules in the activation, proliferation and apoptosis of T cells; 3) Determine whether the suppression of T cell activation and proliferation or T cell apoptosis during filovirus infection can be reversed by induction of DC maturation. The project will involve the infection of DC derived from donor blood with a recombinant EBOV, its mutants with the IFN-I antagonizing domains disabled, or MARV; stimulation of primary human CD4+ and CD8+ T cells; characterization of the effects of filovirus infection and IFN-antagonizing domains on transcriptomes of infected DC and stimulated T cells by deep sequencing; blockade of IFN-I signaling and immuno-suppressive and pro-apoptotic pathways; and attempts to rescue maturation of infected DC with cytokine cocktails. Together, these experiments represent a broad, integrated approach to investigate the mechanisms of disabling the adaptive immune response by filoviruses.