Science. envelope structure, together with an understanding of how bNAbs emerge has guided the design of new immunogens and vaccine strategies that show promise for eliciting protective antibodies. Keywords: HIV Envelope, Broadly Neutralizing Antibodies, Germline-targeting immunogens, Trimer INTRODUCTION HIV-1 is one of the most variable and glycosylated viruses known, making it an especially challenging target for neutralizing antibodies. While almost all infected people develop antibodies to the HIV envelope which have some cross-neutralizing activity (1), only about 20-30% of people develop responses that are considered truly broadly neutralizing (2-5). Furthermore, it generally takes years of infection for these broadly neutralizing antibodies (bNAbs) to evolve, and they often have unusual features not favored by the immune system, including extensive somatic hypermutation (SHM), very long or short CDRs, and autoreactivity (6). This suggests difficult pathways to developing bNAbs naturally, even in the context of ongoing viral replication, and poses challenges to their elicitation by vaccination. Despite this, there is strong rationale for pursuing bNAbs to prevent HIV infection. Passive immunization of bNAbs has long been known to protect non-human primates from infection (reviewed in (6)). Indeed, a recent AZD-3965 AZD-3965 study shows that a single injection of bNAbs protects against repeated exposure for up to 23 weeks (7). Furthermore, much of the accessible part of the HIV trimer is now known to be vulnerable to bNAbs (8). These conserved epitopes include the V2 site, the N332 glycan supersite, the membrane proximal external region (MPER), the CD4 binding site (CD4bs), and the gp120-gp41 interface, most recently shown to include the fusion peptide (9). Elucidation of the native envelope trimer structure (10-16), and of how bNAbs emerge in vivo, has informed the design of new immunogens and vaccine strategies. Some of these immunogens have been tested in non-human primates with promising results, however none have yet broken the barrier to achieving neutralization breadth. This review will cover recent studies that have provided insights into how to overcome these viral and host barriers, and the latest thinking in immunogen design. VIRUS-ANTIBODY CO-EVOLUTIONARY STUDIES PROVIDE A MODEL FOR BREADTH The high levels of SHM of many HIV bNAbs suggest a long co-evolutionary trajectory, requiring variation in both the virus and the antibody. Indeed, two recent studies showed that bNAb lineages evolve at least as fast as HIV, particularly at the early stages of their development, though these rates later decline (17, 18). Though selection and mutability account for some of this decrease (18), the role of viral escape in limiting antibody maturation remains understudied. Nonetheless, this incredibly rapid host evolution provides a mechanism for the extraordinary diversity achieved within long-lived bNAb lineages (17). A key event in the development of breadth is the viral diversification that precedes bNAb emergence (19-21). A detailed study of the V2-targeting CAP256-VRC26 lineage provided a mechanism for how this diversification contributed to breadth, showing that as the antibody lineage matured, some members learned to tolerate diversity that had been created by viral mutations at key sites in the epitope (22). In contrast, dead-end antibodies that were unable to tolerate diversity failed to mature, while other off-track antibodies matured, though not towards breadth (22). In that AZD-3965 study, virus mutations occurred through escape from earlier members of the same lineage, but another explanation for increased viral diversity has come from studies of co-operating antibody lineages Klf2 within single individuals (23, 24). This was first shown in donor CH505, where the CH235 CD4bs lineage drove escape mutations that enhanced neutralization by a second CD4bs lineage, CH103, resulting in breadth. A fascinating follow-up study showed that ultimately the CH235 lineage acquired even greater breadth, highlighting the role of.