Technological Development Component

人类对传染性病原体的免疫反应的研究因以下几个因素而变得复杂：1)许多微生物产生拮抗蛋白，干扰免疫反应的各个方面；2)免疫反应是通过与多种细胞类型的一系列相互作用产生的，单个病原体与每种细胞类型的相互作用可能不同；3)人类群体的遗传变异。技术开发部分（TDC）通过开发新技术来解决这些问题，这些新技术将提供人类对病毒病原体反应的全面概况。虽然很明显，大多数病毒（如果不是全部的话）编码与逃避免疫有关的基因产物，但对这些病毒基因对人类免疫反应的影响知之甚少。我们正计划建立以下检测方法：1)使用表达此类拮抗剂的嵌合I型病毒（CV）识别和表征免疫功能的病毒抑制剂；2)对人体免疫应答在先天免疫、先天适应界面和适应性免疫三个阶段的诱导进行分子描述；通过在I基因转录、IFN信号传导、细胞因子/细胞表面标记物表达和肽呈现水平上表征细胞对病毒感染的反应动力学；3)试图确定影响免疫应答诱导关键基因转录水平的人类基因多态性。利用Newcastles病或仙台病毒作为主干构建CV是TDC的一项整体创新，并且将允许对被认为编码免疫拮抗剂蛋白的单个病原体基因进行安全研究；2)刺激干扰素通路的激活和髓样树突状细胞（mDCs）的成熟，允许读取异源拮抗剂活性，50-100个基因的qPCR小组将用于研究病毒和相关细胞类型在免疫的三个阶段中的相互作用。第1组（先天）用于检查先天免疫

Studies of human immune responses to infectious agents are complicated by several factors: 1) many microbes produce antagonistic proteins that interfere with various aspects of the immune response; 2) immune responses are generated through a series of interactions with multiple cell types and individual pathogens may interact differently with each type; 3) genetic variation in the human population. The Technology Development Component (TDC) confronts these issues through the development of novel technologies that will provide a comprehensive profile of human responses to viral pathogens. Although it has become apparent that most if not all viruses encode gene products implicated in evasion of immunity, very little is known on the impact of these viral genes on the human immune response. We are planning to establish assays that will 1) identify and characterize viral inhibitors of immune function using chimeric I viruses (CV) expressing such antagonists; 2) generate a molecular depiction of the induction of the human immune response against viruses at three stages: innate immunity, the innate-adaptive interface and adaptive immunity; by characterizing the kinetics of the cellular response to viral infection at the levels of I gene transcription, IFN signaling, cytokine/cell surface marker expression and peptide presentation; 3) attempt to identify human gene polymorphisms that impact the levels of transcription of critical genes involved in the induction of the immune response. The construction of CV using Newcastles disease or Sendai virus as a backbone is an integral innovation of the TDC, and will 1) allow safe investigation of individual pathogens genes thought to code for immune antagonist proteins; 2) stimulate interferon pathway activation and maturation of myeloid dendritic cells (mDCs) allowing a readout of heterologous antagonist activity, qPCR panels of 50-100 genes will be used to investigate the interactions of viruses and pertinent cell types at each of the three stages of immunity. Panel 1 (innate) will be used to examine innate immune gene transcription in the three major interferon producing cell types (mDCs, pDCs and epithelial cells). Panel 2 (innate-adaptive) will measure upregulation of genes activated in mDC during maturation and panel 3 (adaptive) will evaluate genes activated in T cells stimulated with allogeneic mDCs. Using samples from the clinical core and wild type or chimeric viruses containing antagonists, we will generate profiles of human responses to viruses and the map the impact of the antagonists. Patient samples will be further tested for genetically determined transcription variation in key immune candidate genes by a simple allelic imbalance assay. This data collected will be correlated with patient medical history and should assist in identification of subgroups of individuals with altered susceptible to specific virus infections. The technology developed by the TDC will be used in active collaboration with the other investigators in the center to identify and investigate viral antagonists. The human cellular immune response profiles to viruses and the impact of viral antagonists will be compiled and analyzed in our database.