Objective. Our objective is to elucidate how neutralizing antibody (nAb) responses against live viruses are generated in vivo within lymph nodes (LNs). To this end we will make use of state-of-the-art imaging technology (i.e. multiphoton intravital microscopy [MP-IVM]), fluorescent replication-competent viruses and dedicated mouse models.Background/Rationale. nAbs are critical for virus control, prevention of re-infection and protection conferred by available vaccines. Thanks to the recent advent of MP-IVM, several cellular and molecular events by which LNs orchestrate humoral immune responses have been clarified. As none of these studies employed live viruses, further work is required to extend these results to a more relevant natural setting. Also, the mechanisms by which cytopathic viruses (e.g. rabies virus in humans and vesicular stomatitis virus [VSV] in mice) induce early high affinity nAb responses while non-cytopathic viruses (e.g. hepatitis C virus in humans and lymphocytic choriomeningitis virus [LCMV] in mice) fail to do so remain poorly understood. Our rationale is based on the notion that, by bringing together unique reagents and advanced technology, we can - at last - address these issues experimentally.Description of the project. The spatial and temporal constraints whereby virus-specific naïve B cells encounter viral antigen, interact with different LN cells and differentiate into plasma cells will be dynamically dissected in the various LN sub-compartments of mice infected with live cytopathic (VSV) and non-cytopathic (LCMV) viruses.Anticipated output. We will provide the first complete in vivo imaging survey of virus-specific B cell activation, from the first minutes of viral entry into LNs to the generation of high affinity nAb-secreting cells. We will also identify virus-induced mechanisms interfering with nAb responses. This new knowledge will provide insight into aspects of viral immunity that may lead to novel rational vaccine strategies.