Improving our understanding of the early immunological responses to influenza A virus (IAV) is significant due to the annual IAV morbidity and mortality, the risk of a catastrophic new pandemic and the limitations of current vaccines and therapeutics. Many aspects of the development of protective immunity and the maintenance of immune homeostasis in the human lung are largely unknown. Our central theme is that the early immune response to the initial IAV infection in human lung is an emergent property from many cell types, host processes, pathogen effects, and microenvironment factors, propagating across scales and interacting in space and time. How stochastic processes and the resulting single cell response variation influence tissue level immunological response is an important question we are poised to address. Predictive immunological modeling, which is needed to understand this complex system, requires a collaborative program to anchor models in detailed human time course immunological data obtained in primary human cells and human lung tissue. Our existing NIAID contract-funded program (PRiME) for modeling immunity for biodefense has studied the dynamic immunological responses of human monocyte-derived dendritic cells (mo-DC) to IAV, providing new insights into host, virus and stochastic mechanisms that operate in mo-DCs following infection. We now propose to advance predictive modeling of IAV infection in humans by significantly expanding our focus to include multiple cell types that interact in space and time--experimentally-validated models will be developed for the human tracheobronchial epithelial cells (HTBE), which are the initial line of defense against virus, and the primary CD1c+ human DC subtype cells which respond to IAV during the first days of infection to contribute to the immediate immune response and to initiate adaptive immunity. Model parameterization and validation are enabled by new key technologies we have established, including barcoded recombinant viruses, single cell assays, human lung explants and multiscale modeling methods. Project I will quantify the responses of fully differentiated primary HTBE to IAV infection. Projec 2 will quantify the responses of primary CD1c+ DC and fresh human lung tissue to IAV infection. Project 3 will develop multiscale models of IAV infection in HTBE and DC in culture and in the context of the lung microenvironment. The wild-type and recombinant viruses studied will be generated in Core B: Virology. The immune assays will be standardized for all experimental projects by Core C: Immune Assay. The data analysis, data handling and dissemination of models and data will be facilitated by Core D: Model and Data Management. Central to all experimental and modeling projects and service cores is Core A: Administrative, which will coordinate all program activities and develop educational programs. This research program will improve the understanding of the mechanisms underlying the immune response to IAV in order to provide the basis for improved strategies for therapeutics and vaccination.
Influenza A virus infection is an important public health threat and current therapeutic and preventative approaches have limitations. We propose a mathematical modeling and experimental collaborative study of the early immunological responses to influenza virus infection in key human cell types and in human lung tissue. The insight gained will further the rational development of improved therapeutics and vaccines for influenza. Project 1- Immunity to Influenza in Primary Lung Epithelial Cells Project Leader: Garcia-Sastre, Adolfo DESCRIPTION (as provided by applicant): In this Project, we will investigate the dynamics of the host innate immune response induced by influenza A virus (IAV) in the main target cells of IAV replication in vivo: the human respiratory epithelial cells. For this purpose, we will compare induction and regulation of host responses upon infection of differentiated human tracheobronchial epithelial cells (HTBEs) with genotypically and phenotypically distinct IAV strains of human clinical relevance. Our project builds upon the previous characterization of these strains in monocyte-derived dendritic cells during our past NIAID contract-funded program for modeling immunity for biodefense (MIB) (PRiME). We now propose to elucidate the mechanisms underlying the immunological responses to IAV of the HTBEs. In Aim 1, the dynamics and special-temporal regulation of the host response induced by seasonal and pandemic H1N1 influenza virus strains will be studied using single cell technologies, such as CyTOF and RNA FISH assays. In concert with mathematical modeling in Project 3, we will test the hypothesis that the innate immune responses and the transcriptional re-programming of HTBEs during IAV infection emerges from the interplay of several factors including differences among single cells, differences among IAV strains, and the autocrine and paracrine responses of the cells in response to the secreted cytokines. In Aim 2, we will determine how the host response varies according to the strength of IAV sensing, and to the ability of IAV to block host responses and to evade antiviral effectors of these responses. This will be done by probing HTBEs with mutant and reassortant IAV specifically differing in one of these parameters. This will also allow the validation (or further refinement) of the predictions of the model generated by Project 3. In Aim 3, we will study the impact on HTBE innate immune responses of clinically relevant inhibitors of influenza virus replication working at different levels during the virus cyce. Responses in HTBEs will be compared with those in primary dendritic cells (studied in Project 2), and validated in a human lung explants model (also studied in Project 2). As the innate immune response in HTBEs is one of the first barriers against IAV infection, a better understanding of the regulation of the dynamics and magnitude of these responses and their impact in virus replication will help to better define what determines the outcome (virus inhibitio versus unimpeded virus replication) of IAV infection.
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