Abstract A major goal of HIV-1 vaccine research is the design of immunogens capable of inducing broadly neutralizing antibodies (bnAbs) that bind to the viral envelope glycoprotein (Env). While many HIV bnAbs have been isolated from chronically infected patients, raising bnAbs by immunization has been difficult. The principal problems appear to be (1) that bnAb epitopes are sub-dominant and so the response must be primed and boosted in such a way as to avoid off-target responses and favor the desired specificities, (2) that most forms of Env bind poorly to unmutated precursors of bnAbs so that bnAb responses are not typically primed appropriately and (3) we do not understand how best to boost responses once primed to yield bnAbs that typically have an unusual amount of somatic mutation. In this proposal, we address these problems by study of engineered immunogens in bnAb knockin mice. We focus on two classes of bnAbs, VRC01 and PGT130, which we predict will be readily raised by vaccination in humans using appropriately engineered immunogens. VRC01 is the prototype bnAb of a class that recognizes the CD4 binding site (CD4bs) and PGT130 is a representative of a class of glycan-dependent bnAbs that bind the V3 loop and mannose patch on Env. Both antibody classes have been described in many HIV infected individuals and are particularly potent and broad suggesting that a vaccine able to elicit such Abs would provide protection at relatively low Ab concentrations. We have previously generated mice engineered to carry B cells with germline reverted (gl)-VRC01 immunoglobulin heavy (H) and light (L)-chains, and here we propose to generated new knock-in mice carrying gl-PGT130 H- and L-chain genes. The mice express the B cell receptors that should be triggered and matured to generate VRC01 or PGT130 class bnAbs and will be used to investigate immunogens and immunization strategies. For priming, we will use existing germline-targeting immunogens reactive to gl-VRC01 and we will generate novel engineered immunogens targeting gl- PGT130 and. For boosting we will test a range of engineered and natural immunogens to focus and enhance intermediate and final steps in bnAb maturation. This study will test the generality of our germline-targeting approach, discover new vaccine candidates, and teach us lessons on how to maximize subdominant anti-viral responses that should inform human vaccination strategies in general.