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.
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