Monocional Antibody and Protein Core

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The approach used in this HIVRAD application for the development of HIV candidate vaccines is based on a reverse vaccinology strategy which uses human monoclonal antibodies (mAbs) with broad and potent anti-viral activity to identify cognate epitopes on the virus envelope (Env) and engraft these structures into scaffold proteins to be used as immunogens. These epitope-scaffold immunogens are then used in vivo to elicit Abs with anti-viral activities similar to those displayed by the parental mAbs. The proposed work for Core B is divided into two specific aims: Aim 1. Production of human mAbs generated in Projects 1 and 2. In this aim, we will scale up production of human mAbs generated in Projects 1 and 2 on the basis of their specificity for V2 or for V2/V3 quaternary neutralizing epitopes (QNEs), and their ability to mediate a variety of anti-viral functions. We will produce sufficient quantities of mAbs to perform extensive immunochemical, functional, and crystallographic studies as required by the work scopes of Projects 1 and 2. These mAbs will be produced by 293 cells transfected with plasmids containing the heavy and corresponding light chain genes of selected mAbs which will have been amplified from antigen-specific B cells. Aim 2. Protein production for Project 1, 2 and Core C. In this aim, we will produce: a) biotin-tagged recombinant epitope-scaffold proteins for staining antigen-specific B cells to be selected for mAb development by molecular methods in Projects 1 and 2, and b) epitope-scaffold proteins to be used as antigens in Projects 1 and 2 and as immunogens in Animal Studies Core (Core C). These epitope-scaffold proteins will be produced primarily in 293 human embryonic kidney cells, or alternatively in other mammalian or insect cells. Production in eukaryotic cells will support glycosylation which is needed to preserve the conformational epitopes of V2 and the V2/V3 QNEs, while production in ¿ coli may be useful for the study of linear epitopes. Our extensive experience in the development of human anti-HIV-1 mAbs and production of recombinant scaffolded proteins ensures the high quality of these reagents which are essential for the successful completion ofthe proposed HIVRAD work scope.