DC-SIGN is a calcium-dependent lectin that binds avidly to HIV Env gpl20 and can transfer bound virions to permissive cells such as CD4+ lymphocytes. This observation coupled with its expression on submucosal dendritic cells (DCs) has led to the suggestion that DC-SIGN may play a sentinel role in the sexual transmission of HIV by serving as the conduit for the transfer of HIV from the mucosa to secondary lymphoid organs. This transfer of virus is hypothesized to be a crucial step in the establishment of a primary HIV infection. Since DC-SIGN binds to specific carbohydrate structures on the viral Env gpl20, and carbohydrate structures are by themselves not very immunogenic, a protein-based immunogen which mimics the DC-SIGN binding glycan structures on gpl20 can potentially elicit antibodies which can block gp 120/DC-SIGN binding and thus help in preventing the establishment of a primary HIV infection. This proposal describes a novel strategy to obtain peptide-mimics of DC-SIGN-binding glycan structures on gpl20 using phage-display technology. This strategy involves panning a multitude of peptide-displayed libraries, including """"""""landscape"""""""" libraries on cell surface-expressed DC-SIGN. We describe elution and panning strategies, which not only overcome the background inherent in cell surface pannings, but also increase the probability of obtaining bona fide mimics of DC-SIGN binding glycan structures on gpl20 (Aim 1). Candidate phage clones have already been identified. Next, we will biochemically characterize the peptide mimics to determine the best candidates for follow-up immunogenicity studies (Aim 2). We will subsequently determine the immunogenicity of these peptide mimics and assess whether they can indeed elicit antibodies that can block DC-SIGN/gpl20 interactions (Aim 3). This proposal has the added advantage that in the event that none of the peptide mimics selected elicit gpl20-cross-reactive antibodies, the peptide mimics could still be used as lead candidates for microbicide development. Thus, these peptide mimics can be followed up in a vaccine or microbicide strategy to target the initial attachment stage of HIV to dendritic cells, and can be used as reagents to elucidate pathogenic mechanisms involved in dendritic cell mediated viral transfer. Therefore, we believe this research proposal appropriately meets the research objectives defined by the Innovation Grant Program for AIDS Research (PA-02-046). ? ? ? ?
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