The overall objective is to develop immunogens that will initiate and select HIV-1 broad neutralizing antibody (bnAb) lineages directed to the distal membrane proximal external region (MPER) of HIV Env gp41. Distal gp41 MPER antibody types such as 10E8 and DH511 are desirable because they are among the most broad and potent bnAbs isolated. There are two strategies for bnAb immunogen design lineages. (1) Define bnAb clonal lineage genealogies, infer the bnAb unmutated common ancestor (UCA) and select autologous Envs that bind?termed B cell lineage immunogen design. (2) Structural-based design, using structures of sequential lineage Abs to design Envs that bind to bnAb lineage members. Here we propose to combine the strengths of both strategies to design immunogens that can initiate and induce distal MPER bnAbs. We will use the newly isolated DH511 lineage UCA, intermediate antibodies (IAs) and bnAbs as reagents upon which to design sequential immunogens that will select DH511-like precursors and lead to bnab development (Project 1, William Schief, PI), and to test these immunogens in physiologically relevant knock-in mouse model of bnAb development (Project 2 (Munir Alam, PI, Small Animal Models Core, Ming Tian, PI, Fred Alt, Co-I). A computational program, Antigen Receptor Mutation Analyzer for Detection of Low Likelihood Occurrences (ARMADiLLO) (Project 2) that allows for definition of the critical antibody somatic mutations to be induced will be used to determine key IAs that a successful vaccine will need to target. Overall Specific Aim 1. Define the key IAs and antibody somatic mutations that a successful vaccine will need to select to lead to distal MPER bnAb induction. (Projects 1 and 2) Overall Specific Aim 2. Design of germline targeting (GT) prime and boost immunogens that bind to the DH511 precursors and to key IAs and mature DH511 bnAbs in optimal affinities and can select the correct/desired IAs and bnAbs. (Project 1) Overall Specific Aim 3. Solve co-crystal and cryoEM structures of DH511 and DH511-like lineage antibodies with Env immunogens that move the lineage along the bnAb maturation pathway and enable design of additional immunogens to complete the induction of distal MPER bnAbs B cell lineages. (Project 1) Overall Specific Aim 4. Selection of optimal Env immunogens from immunizations of DH511 UCA and IA VH and VL knock-in mice. This will be accomplished by use of the novel DH511 UCA VHDJH-rearranging mouse recently developed by Ming Tian and Fred Alt at Harvard. (Small Animal Core; Projects 1 and 2). This collaboration of three leading academic teams in HIV vaccine immunogen design will bring together expertise in structure-based and lineage-based design, and will be a powerful approach to the problem of vaccine induction of disfavored antibody lineages in general and distal MPER bnAbs in particular.