Elicitation of cross-neutralizing Abs (cNAbs) to inhibit entry of diverse clinical HIV isolates following Env vaccination remains a high priority to develop a broadly effective vaccine, but the elicitation of cNAbs against cross-conserved Env determinants has been elusive. However, very recently we accomplished an important initial step in this process. We primed rabbits with immunization of native flexibly linked (NFL) trimer-liposomes containing targeted N-glycan deletions proximal to the highly conserved CD4 binding site (CD4bs) to better activate B cell responses to this region. The engineered gaps in the N-glycan shield were gradually restored by heterologous sequential boosting. We elicited cross-neutralizing activity in the serum and purified IgG of selected animals and, subsequently, cloned two cNAbs. The cNAb most relevant to this proposal, E70, provides proof-of- principle for a fundamentally different approach. The CD4bs-directed E70 is relatively potent against multiple tier 2 isolates and, importantly, recognizes approximately 50% of N-glycan as part of its epitope as well as adjacent conserved polypeptide that is part of the CD4bs. (The 2nd mAb, 1C2, is a broadly neutralizing antibody (bNAb), neutralizing 80% of a 40 isolate panel and is directed to the gp41:gp120 interface.) The high-resolution cryoEM structure of the E70 epitope on the NFL native-like trimer provides critical preliminary data on how to train the immune system to recognize other N-glycans proximal to the conserved protein surface of the CD4bs to mediate cross-neutralization of tier 2 isolates. We call this approach “N-glycan baiting”. For this approach, we will leave one selected N-glycan proximal to the CD4bs intact as “the bait” while removing all other proximal N- glycans on the initial priming immunogens. The priming will be performed at 6 individual N-glycan sites that ring the conserved CD4bs protein surface. This allows a given cell receptor to recognize chimeric epitopes comprised of the “anchor glycan” and surrounding conserved polypeptide. With boosting, all N-glycans are eventually restored in a step-wise manner, limiting angles of approach to a given N-glycan bait to regenerate the intact N- glycan shield while still driving a subset of B cells directed to the original anchor glycan and protein as a chimeric, non-self-epitope. This strategy fundamentally differs from our original approach in targeting the receptor binding site by full deletion of all proximal N-glycans. Accordingly, the major objective of this grant is to generate novel HIV trimeric Env immunogens by structure-based design, containing N-glycan deletions at the CD4bs but to retain individual “anchor glycans”. We will first characterize the N-glycan-anchored priming immunogens by state-of-the-art biophysical methods (Aim 1). Next, we will determine immunogenicity in small animals in Aims 2 and 3, including neonates. Following targeting of the N-glycan shield in small animals, we will perf...