Mechanisms of "immune paralysis" caused by filoviruses

埃博拉丝状病毒（EBOV）和马尔堡病毒（MARV）感染导致“免疫麻痹”，其特征在于T细胞应答缺陷和淋巴细胞减少，尽管缺乏T细胞感染，以及死亡率高达80%的严重出血热。在过去的十年中，研究人员确定了由丝状病毒蛋白介导的I型干扰素（IFN-I）拮抗作用的多种机制。然而，丝状病毒快速和有害地使适应性免疫应答失能的机制仍然未知。 树突状细胞（DC）是专职的抗原呈递细胞，被认为是容易被感染的，但不经历正常的成熟。我们的初步数据已经证明了由EBOV引起的IFN-I拮抗作用与缺乏DC成熟之间的强烈联系。该提议的中心假设是，由丝状病毒蛋白和多种机制介导的IFN-I应答的异常强烈和冗余的拮抗作用阻断了DC的成熟，导致T细胞的缺陷和/或异常刺激。该假说将通过追求三个具体目标来检验：1）确定IFN拮抗结构域和IFN-1信号传导在DC成熟和T细胞活化、增殖和凋亡中的作用; 2）确定由感染的DC分泌的可溶性因子和特异性抑制和促凋亡分子在T细胞活化、增殖和凋亡中的作用; 3）确定丝状病毒感染期间T细胞活化和增殖或T细胞凋亡的抑制是否可以通过诱导DC成熟来逆转。该项目将涉及用重组EBOV、其IFN-I拮抗结构域失活的突变体或MARV感染来源于供体血液的DC;刺激原代人CD 4+和CD 8 + T细胞;通过深度测序表征丝状病毒感染和IFN拮抗结构域对感染的DC和刺激的T细胞的转录组的影响;阻断IFN-I信号传导和免疫抑制和促凋亡途径;并尝试用细胞因子混合物挽救受感染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应答失能的机制。

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.