The induction of broadly cross-neutralizing antibodies (Abs) that can protect healthy individuals against HIV infection remains a major challenge for vaccine development. These neutralizing Abs are observed in the course of natural HIV-1 infection, appearing 2 to 3 years post infection raising the question of whether or not they can be induced during a relatively short period of immunization. We propose a new approach to (a) employ an immunogen (mimotope-fusion protein) which targets selected immunoglobulin (Ig) gene-encoded Abs on naive B cells that are the precursors of anti-V3 neutralizing Abs and (b) monitor for an affinity maturation and development of cross-neutralization potency of pseudoviruses. Toward these goals, we have developed a rationally designed immunogen based on VH5-51 mimotope that mimics the highly conserved V3 epitopes recognized by human cross-neutralizing anti-V3 monoclonal Abs (mAbs) encoded by a pairing of the VH5-51 and VL lambda genes. We hypothesize that a VH5-51 mimotope can be targeted to macaque B cell receptors (encoded by the VH5-51 and VL lambda genes), where it will induce Abs with significantly enhanced affinity maturation compared to the control mimotope (non-VH5-51), which will induce anti-V3 Abs encoded by different Ig genes. In the first aim, we will generate monoclonal anti-V3 Abs from antigen-specific single B cells derived from rhesus macaques immunized with two immunogens to specifically elicit anti-V3 Abs encoded by the VH5-51 or by other non-VH5-51 genes. Two groups, each comprising three rhesus monkeys, will be immunized with gp160 DNA prime in combination with a VH5-51 mimotope-CTB fusion protein or gp160 DNA prime with control non-VH5-51 mimotope-CTB. Blood specimens from each animal will be drawn at pre-immunization, during immunization and at 1, 3, 6, 12 and 18 months post-last immunization. The longitudinal PBMC specimens from one animal in each group will be chosen for production of anti-V3 mAbs from IgG+ single B cells selected using the biotinylated V3-Fc fusion protein. The Ig variable genes from V3-specific B cells will be amplified by RT-PCR, cloned into expression vectors, and full-length IgG mAbs will be produced from 293T cells upon plasmid co-transfection. In the second aim, we will monitor the maturation of anti-V3 mAbs, sequentially produced from macaques immunized with the VH5-51 and non-VH5-51 immunogens by measuring mutation rates, relative affinity (50% maximal binding) and neutralizing activity (IC50). The profile of changes in the affinity and neutralizing activities wil be compared between VH5-51- and non-VH5-51-derived anti-V3 mAbs. Results that demonstrate the feasibility of targeting the particular Ig gene-encoded V3 B cell receptor would provide opportunities to target other Ig genes encoding neutralizing Abs with different specificities. For vaccine development, the immunogen based on a Ig gene-targeted mimotope can be used for combined boosting with gp120 to spike the immune response against particular envelope neutralizing epitope.