Antibody production was performed using a previously described protocol (3,81). intra-HCDR3 disulfide. The mechanism of viral escape was examined via Cbz-B3A single-genome amplification/sequencing and glycan mutations around the N332 supersite. Our findings further emphasize the V3 glycan supersite as a prominent target for Env-based vaccine design. == INTRODUCTION == During HIV-1 infection, a small fraction of individuals mount effective antibody responses against coevolving viruses, which eventually become broadly neutralizing antibodies (bNAbs) capable of countering diverse Cbz-B3A HIV-1 isolates with exceptional potency (13). Most of the known HIV-1 bNAbs have unusual characteristics such as selective germline gene usage, high degree of somatic hypermutation (SHM), and long heavy-chain complementarity-determining region 3 (HCDR3) loops. Precursors of bNAbs may emerge in the early stage of infection and later achieve neutralization breadth and potency through an extensive maturation process (47). Such bNAb precursors often display low levels of autologous neutralization but can initiate the process of recognizing conserved epitopes on the envelope glycoprotein (Env) in response to a constantly evolving viral population (5,6,8). Multiple bNAb epitopes on HIV-1 Env have been identified, such as the CD4 binding site (CD4bs), glycan-rich epitopes at the V2 apex and V3 base, gp120-gp41 interface, fusion peptide, and membrane-proximal external region (MPER) (9), in addition to the silent face (10,11). HIV-1 cohort studies found ~15% of individuals with broad serum neutralizing activity and nearly half of these broad neutralizers showing bNAb responses to the oligomannose-rich patch centered Rabbit polyclonal to VDAC1 at Asn332 (12,13), suggesting that the immune system can effectively recognize this glycan-rich V3 epitope on the Env spike. Panels of human antibodies to this site have been isolated that exhibit moderate [PCDN lineage (6)] to high potency [PGT121 (13), PGT128 (14,15), 101074 (16), and BG18 (2)], as well as substantial breadth (28 to 68%) (17). These bNAbs penetrate the glycan shield of the Env spike usually with their light-chain CDR1/2 (LCDR1/2), HCDR2, and long HCDR3 loops and interact with both oligomannose glycans and peptide components of the N332/V3 supersite. Furthermore, structural studies (1821) have revealed various angles of approach by which these bNAbs access the CCR5 coreceptor binding motif, GDIR (22,23). Env trimerbased immunogens RC1 (24) and N332-GT (25) were designed to elicit human bNAb precursor antibodies targeting the N332/V3-glycan patch. It has been reported that a common mechanism used by HIV-1 to escape the N332/V3-specific bNAbs is through mutations of Asn332(6), Asn295, and Asn301(26) that eliminate N-linked glycans; a glycan shift from position 332 to 334 (4,27); or an unusual elongation of the V1 loop with additional glycans and disulfide bonds (28). However, some HIV-1infected individuals can develop broad serum neutralization against the N332/V3 supersite by switching the glycan from position 334 to 332 (27). It is, therefore, imperative to study more elite donors to understand how bNAbs recognize the shifting glycans at or around the V3 base and how the coevolving viruses counter Cbz-B3A it. One of the key questions is whether any other features could be used by bNAbs to enhance the glycan epitope recognition and, hence, HIV-1 neutralization, in addition to diverse angles of approach and long CDR loops. In this study, we examined an N332/V3-directed bNAb response in an HIV-1 subtype Binfected Chinese donor, CBJC438. This individual showed sustained plasma neutralizing activity over a period of 8 years from 2005 to 2012. Using a heptad repeat 1 (HR1)stabilized BG505 trimer (29) as bait, we isolated two monoclonal antibodies (mAbs) (438-B11 and 438-D5) from the 2008 sample that exhibited exceptional neutralizing potency with moderate breadth. To understand how these two related bNAbs recognize and neutralize HIV-1, we determined their structures in the unbound form and in complex with an uncleaved prefusion-optimized (UFO) BG505 Env trimer. Crystal structures revealed how these bNAbs approach the N332/V3 supersite on the trimer with a disulfide-locked HCDR3 loop and a unique angle of approach relying on heavy chaindependent recognition. Longitudinal repertoire analysis uncovered a diverging bNAb lineage that expanded.