Project Summary/Abstract It is believed that broadly neutralizing antibodies (bNAbs) that bind to the HIV-1 Envelope glycoprotein (Env) will be an important component of an immune response elicited by an effective HIV-1 vaccine. However, efforts to elicit bNAbs through vaccination with recombinant Env have not yet been successful. One of the critical barriers to eliciting bNAbs is thought to be the failure of recombinant Env proteins to bind to B cell receptors (BCRs) on naive B cells that ultimately give rise to bNAbs. In an attempt to overcome this barrier, this project aims to generate and test the ability of novel, non-Env immunogens that are highly specific for bNAb precursor BCRs. To generate these immunogens, we will immunize mice with germline bNAb precursors targeting multiple, distinct epitopes on Env. We will then employ a high-throughput screening platform to isolate murine antibodies that are highly specific for the paratope (or idiotype) of the bNAb precursor BCRs. These ?anti- idiotypic? antibodies will be evaluated for their ability to seek out, and stimulate bNAb precursor B cells using complementary ex vivo and in vivo approaches. We will use fluorescently labeled anti-idiotype antibodies to sort B cells from PBMC samples obtained from healthy donors, which will allow us to determine whether these anti-idiotypic antibodies can selectively bind to putative bNAb precursor B cells in humans. We will also evaluate the ability of multimeric anti-idiotype derivatives to stimulate and expand bNAb precursor B cells in a humanized mouse model, and test whether these expanded B cells can be guided along the pathway to becoming neutralizing by boosting with recombinant Env.