Many pathogens enter via the mucosa, including such NIAID Category A, B, and C pathogens as influenza, Bacillus anthracis, Francisella tularemia, and Yersinia pestis. There is currently no reliable way to assess the mucosal immune response to invading pathogens or to vaccines against them. Our goal is to develop technology to better characterize mucosal B cell responses induced by DC-targeting vaccines. Strategies will include: 1) transformation of mucosal B cells using the EBV/CD40 system and 2) isolation of Ig mRNA from single cells. AIM 1 will establish methods to sort antigen-specific B cells from blood and single cell suspensions from mucosa. We will develop reagents to isolate HA-specific B cells from the blood of vaccinated healthy subjects. The detection is via the surface B cell receptor and based on the direct labeling with B cell tetrameric antigens using technology established by our collaborator, Dr. B. Haynes, for HIV antigens. AIM 2 will demonstrate that the sorted B cells are antigen-specific by: 1) the EBV/B cell transformation system, which allows immortalization and cloning of human memory B cells and 2) isolation of Ig mRNA from single cells to assess the antibody repertoire of plasmablasts and plasma cells. PCR from cDNA is used then to amplify VH and VL sequences for cloning and expression in 293 cells as secreted IgG. The recombinant antibodies are tested for their specificity and affinity using our recently developed multiplex technology, which permits assessment of a large spectrum of antigens presented on beads. When validated with blood cells, the feasibility of this strategy will be tested using single-cell suspensions obtained from mucosal sites from human subjects (Core C and D), humanized mice (Project 3), and non-human primates (Project 4). AIM 3 will establish a methodology to isolate antigen-specific B cells from mucosal tissue sections. We will use labeled antigen with co-staining for CD138 and k/l to locate antigen-specific B cells in frozen tissue sections. This will permit isolating single antigen-specific B cells using laser microdissection (Arcturus). RNA is prepared and PCR from cDNA is used to clone and express corresponding recombinant antibodies as in Aim 2 to determine the antigen specificity of the isolated cells.