New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, ...isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1). Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
HIV's envelope glycoprotein (Env) is the sole target for neutralizing antibodies. The structures of many broadly neutralizing antibodies (bNAbs) in complex with truncated Env subunits or components ...have been reported. However, their interaction with the intact Env trimer, and the structural determinants that underlie neutralization resistance in this more native context are less well understood. Here we use hydrogen/deuterium exchange to examine the interactions between a panel of bNAbs and native-like Env trimers (SOSIP.664 trimers). Highly potent bNAbs cause only localized effects at their binding interface, while the binding of less potent antibodies is associated with elaborate changes throughout the trimer. In conjunction with binding kinetics, our results suggest that poorly neutralizing antibodies can only bind when the trimer transiently samples an open state. We propose that the kinetics of such opening motions varies among isolates, with Env from neutralization-sensitive viruses opening more frequently than Env from resistant viruses.
A major advance in the search for an HIV vaccine has been the development of a near-native Envelope trimer (BG505 SOSIP.664) that can induce robust autologous Tier 2 neutralization. Here, potently ...neutralizing monoclonal antibodies (nAbs) from rabbits immunized with BG505 SOSIP.664 are shown to recognize an immunodominant region of gp120 centered on residue 241. Residue 241 occupies a hole in the glycan defenses of the BG505 isolate, with fewer than 3% of global isolates lacking a glycan site at this position. However, at least one conserved glycan site is missing in 89% of viruses, suggesting the presence of glycan holes in most HIV isolates. Serum evidence is consistent with targeting of holes in natural infection. The immunogenic nature of breaches in the glycan shield has been under-appreciated in previous attempts to understand autologous neutralizing antibody responses and has important potential consequences for HIV vaccine design.
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•HIV neutralizing antibodies were isolated from rabbits immunized with BG505 SOSIP.664•These antibodies target a hole in the glycan shield of BG505•Serum neutralization specificity maps to the same immunodominant glycan hole•Most HIV strains lack a conserved glycan site that could be a neutralization target
This study describes monoclonal rabbit antibodies elicited by the HIV immunogen BG505 SOSIP.664. Previous reports showed high-titer serum neutralization against the BG505 virus, but the epitope remained elusive. McCoy et al. show the neutralizing epitope is a breach in HIV’s glycan shield and suggest other HIV strains contain similar holes.
A highly glycosylated, trimeric envelope glycoprotein (Env) mediates HIV-1 cell entry. The high density and heterogeneity of the glycans shield Env from recognition by the immune system, but ...paradoxically, many potent broadly neutralizing antibodies (bNAbs) recognize epitopes involving this glycan shield. To better understand Env glycosylation and its role in bNAb recognition, we characterized a soluble, cleaved recombinant trimer (BG505 SOSIP.664) that is a close structural and antigenic mimic of native Env. Large, unprocessed oligomannose-type structures (Man8-9GlcNAc2) are notably prevalent on the gp120 components of the trimer, irrespective of the mammalian cell expression system or the bNAb used for affinity purification. In contrast, gp41 subunits carry more highly processed glycans. The glycans on uncleaved, non-native oligomeric gp140 proteins are also highly processed. A homogeneous, oligomannose-dominated glycan profile is therefore a hallmark of a native Env conformation and a potential Achilles’ heel that can be exploited for bNAb recognition and vaccine design.
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•Native-like, cleaved HIV-1 Env mimetics are dominated by underprocessed N-glycans•In contrast, non-native uncleaved trimers undergo greater glycan processing•The Env quaternary structure dictates the degree of glycan processing that can occur•The abundance of homogeneous oligomannose glycans is promising for vaccine design
The HIV-1 envelope spike is the major target for vaccine design. Pritchard et al. show that the quaternary structure of this trimeric glycoprotein determines the processing of its extensive glycan coat. Structural constraints inhibit cellular processing, leaving a largely homogeneous oligomannose population that can be targeted by the immune system.
All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a ...unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151–PGT158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. Because PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer.
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•PGT151 binds an interprotomer epitope formed by gp41 and gp120 on cleaved Env trimers•The PGT151 epitope does not overlap with any other epitope described so far•PGT151 enables isolation of functional cleaved Env from the cell membrane•Membrane-extracted and soluble SOSIP.664 Env trimers are structurally similar
PG9 is the founder member of an expanding family of glycan-dependent human antibodies that preferentially bind the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralize the ...virus. Here, we show that a soluble SOSIP.664 gp140 trimer constructed from the Clade A BG505 sequence binds PG9 with high affinity (∼11 nM), enabling structural and biophysical characterizations of the PG9:Env trimer complex. The BG505 SOSIP.664 gp140 trimer is remarkably stable as assessed by electron microscopy (EM) and differential scanning calorimetry. EM, small angle X-ray scattering, size exclusion chromatography with inline multiangle light scattering and isothermal titration calorimetry all indicate that only a single PG9 fragment antigen-binding (Fab) binds to the Env trimer. An ∼18 Å EM reconstruction demonstrates that PG9 recognizes the trimer asymmetrically at its apex via contact with two of the three gp120 protomers, possibly contributing to its reported preference for a quaternary epitope. Molecular modeling and isothermal titration calorimetry binding experiments with an engineered PG9 mutant suggest that, in addition to the N156 and N160 glycan interactions observed in crystal structures of PG9 with a scaffolded V1/V2 domain, PG9 makes secondary interactions with an N160 glycan from an adjacent gp120 protomer in the antibody–trimer complex. Together, these structural and biophysical findings should facilitate the design of HIV-1 immunogens that possess all elements of the quaternary PG9 epitope required to induce broadly neutralizing antibodies against this region.
The HIV envelope (Env) protein gpl20 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to ...interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man 9 at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully g ly cosy la ted gpl20 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short ß-strand segment of the gpl20 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.
Abstract The need for an effective vaccine to prevent the global spread of human immunodeficiency virus type 1 (HIV-1) is well recognized. Passive immunization and challenge studies in non-human ...primates testify that broadly neutralizing antibodies (BrNAbs) can accomplish protection against infection. In recent years, the introduction of new techniques has facilitated the discovery of an unprecedented number of new human BrNAbs that target and delineate diverse conserved epitopes on the envelope glycoprotein spike (Env). The epitopes of these BrNAbs can serve as templates for immunogen design aimed to induce similar antibodies. Here we will review the characteristics of the different classes of BrNAbs and their target epitopes, as well as factors associated with their development and implications for vaccine design.
Many broadly neutralizing antibodies (bnAbs) against HIV-1 recognize and/or penetrate the glycan shield on native, virion-associated envelope glycoprotein (Env) spikes. The same bnAbs also bind to ...recombinant, soluble trimeric immunogens based on the SOSIP design. While SOSIP trimers are close structural and antigenic mimics of virion Env, the extent to which their glycan structures resemble ones on infectious viruses is undefined. Here, we compare the overall glycosylation of gp120 and gp41 subunits from BG505 (clade A) virions produced in a lymphoid cell line with those from recombinant BG505 SOSIP trimers, including CHO-derived clinical grade material. We also performed detailed site-specific analyses of gp120. Glycans relevant to key bnAb epitopes are generally similar on the recombinant SOSIP and virion-derived Env proteins, although the latter do contain hotspots of elevated glycan processing. Knowledge of native versus recombinant Env glycosylation will guide vaccine design and manufacturing programs.
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•HIV envelope glycans are central features of broadly neutralizing antibody epitopes•Recombinant mimetics of the HIV virus trimer are vaccine candidates•Glycosylation of a leading trimer immunogen is similar to that of infectious virus
Struwe et al. present site-specific analyses of N-glycosylation sites on HIV-1 envelope glycoproteins from an infectious virus and a recombinant trimer mimic. The structural and antigenic details of the glycan shield will be valuable for designing next-generation immunogens and understanding virus neutralization by broadly active antibodies.
The HIV envelope glycoprotein (Env) trimer undergoes receptor-induced conformational changes that drive fusion of the viral and cellular membranes. Env conformational changes have been observed using ...low-resolution electron microscopy, but only large-scale rearrangements have been visible. Here, we use hydrogen-deuterium exchange and oxidative labeling to gain a more precise understanding of the unliganded and CD4-bound forms of soluble Env trimers (SOSIP.664), including their glycan composition. CD4 activation induces the reorganization of bridging sheet elements, V1/V2 and V3, much of the gp120 inner domain, and the gp41 fusion subunit. Two CD4 binding site-targeted inhibitors have substantially different effects: NBD-556 partially mimics CD4-induced destabilization of the V1/V2 and V3 crown, whereas BMS-806 only affects regions around the gp120/gp41 interface. The structural information presented here increases our knowledge of CD4- and small molecule-induced conformational changes in Env and the allosteric pathways that lead to membrane fusion.
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•CD4 binding disrupts quaternary interactions at the Env trimer apex•CD4 binding induces dynamic changes within portions of gp41•NBD-556 and BMS-806 have drastically different effects on Env•Different glycosylation profiles have little effect on Env’s conformational dynamics
The HIV envelope glycoprotein (Env) drives fusion of the viral and cellular membranes by undergoing extensive receptor-induced conformational changes. Guttman et al. probe how each region of Env responds to receptor binding, revealing the allosteric networks in the initial steps of Env activation.
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