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Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env

Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env ARTICLE Received 11 Aug 2014 | Accepted 15 Dec 2014 | Published 5 Feb 2015 DOI: 10.1038/ncomms7144 Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env 1 2 3 2,4 3 Miklos Guttman , Albert Cupo , Jean-Philippe Julien , Rogier W. Sanders , Ian A. Wilson , 2 1 John P. Moore & Kelly K. Lee 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. 1 2 Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195, USA. Weill Cornell Medical College, New York, New York 10021, USA. Department of Integrative Structural and Computational Biology, International AIDS Vaccine Initiative Neutralizing Antibody Center, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA. Department of Medical Microbiology, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands. Correspondence and requests for materials should be addressed to K.K.L. (email: [email protected]). NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ighly potent, broadly neutralizing antibodies (bNAbs) are (CD4bs), its specific binding footprint is not identical to that of 8,21 now being used to guide the design of an effective HIV-1 CD4, which restricts its ability to target the native trimer . 1–3 Hvaccine . One major obstacle in eliciting a protective, More recently identified bNAbs, such as VRC01 (ref. 22) and broad humoral immune response has been the high antigenic PGV04 (ref. 8), more precisely target the CD4bs and have diversity among circulating isolates. The hypervariable loops significantly greater breadth and potency. present on the surface of the envelope glycoprotein (Env) trimer CD4 binding leads to large-scale conformational changes induce antibodies that are highly isolate-specific and generally within Env that create a CD4-induced (CD4i) epitope on gp120, lack breadth (as defined by the percent of HIV-1 isolates the target of some of the earliest known NAbs . One such they can neutralize). The more conserved regions of Env antibody, 17b, recognizes a portion of the bridging sheet that are masked within the trimer before its activation, or are becomes exposed after CD4 binding; its epitope overlaps with the shielded by dense glycosylation; both of these factors restrict the conserved co-receptor binding site . Although their epitopes are 4–7 generation of broadly active responses . Despite these viral conserved, CD4i antibodies such as 17b generally neutralize defenses, a subset of HIV-1-infected individuals can, over time, only lab-adapted or abnormally sensitive primary viruses develop potent (as defined by their IC values in neutralization (‘tier 1’ isolates) on which the co-receptor binding site is 25,26 assays) bNAbs that, in some cases, neutralize 490% of relatively exposed . 2,8 circulating strains . Env glycans are also now understood to be wholly or partially Structural studies have revealed much about the overall involved in the epitopes for several, particularly potent bNAbs. architecture of the Env trimer in its closed, pre-fusion form, Antibody 2G12 recognizes a conserved patch of high-mannose including the arrangement of the variable loops at the trimer apex glycans on the periphery of the CD4bs and has moderate 9–12 27–29 (Fig. 1a) . In this conformation, the V3 region is sequestered neutralization potency and breadth . The more potent PG9/ under the V1/V2 loop structure, while elements of the bridging PG16 bNAbs target the V1/V2 structure at the trimer apex via a sheet are involved in quaternary contacts below the V1/V2 loops; glycan at N160 (refs 30,31). Several bNAbs such as PGT123 that together, these interdomain interactions lead to the occlusion of interact with the base of V3, including glycans at N301 and N332, the conserved V3 and bridging sheet elements that are are among the most potent yet identified . 13–15 components of the co-receptor binding site . Binding of the The different orientations in which many of these antibodies primary receptor, CD4, induces major structural changes within bind various native-like forms of the Env trimer have been Env, including the dissociation of contacts between V1/V2 and described at low to intermediate resolution, via electron 11,17,18,32–34 V3 at the trimer apex and the coalescence of the separate microscopy . However, critical details of how they 4,16–19 elements of the co-receptor binding site . bind the trimer and, by extension, neutralize the virus remain to Defining how antibodies recognize their epitopes on simple or be understood. Although antibody binding is often sufficient to more complex forms of Env has also shed light on how the trimer impede the interaction of a trimer with CD4 or the co-receptor, functions, and how it resists neutralization (Fig. 1b). Antibody other neutralization mechanisms may involve antibodies locking b12 was among the first to be generally accepted as broadly the trimer in the closed, pre-fusion state or, alternatively, driving 20 35,36 neutralizing . Although it recognizes the CD4 binding site its destabilization and inactivation . PG9 17b 2G12 PGT123 V1/V2 CD4, b12, VRC01, V4 PGV04 V5 VRC01 PGV04 b12 75 PG9 Inner PGT123 domain HR2 (gp41) 2G12 17b Outer domain V3 Bridging sheet HR1 (gp41) Layer 1 Layer 2 Layer 3 β-Sandwich domain 0.01 0.1 1 10 Potency –1 median IC (μg ml ) Figure 1 | Neutralizing antibody targets on Env trimers. (a) The structural elements of gp120 are coloured on one protomer of the closed, pre-fusion Env trimer (PDB accession code: 4NCO) .(b) The epitopes of the various NAbs used in this study are highlighted on the structure of the closed, pre-fusion 8,21,22 trimer. The CD4bs, the target of antibodies b12, VRC01 and PGV04 is shown in orange . Glycans at N301 and N332 along the base of V3 make 41 31 up the PGT123 epitope (magenta) . The glycan at N160 and nearby residues in V1/V2 constitute the PG9 epitope (cyan) . Antibody 2G12 recognizes the cluster of glycans at positions N295, N332, N392, N386 and N448 (green) . Antibody 17b sees the CD4i epitope that is only formed and exposed after CD4 binding . The viral membrane is shown at the base to illustrate the orientation of Env (electron microscopy databank (EMD) accession code: 9  1 5022) .(c) The breadth (by per cent of viruses neutralized at concentrations o50mgml ) and neutralization potency (median IC values of the viruses neutralized) are plotted for each NAb. Neutralization data were obtained from BNAber.org . 2 NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Breadth % Viruses neutralized NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ARTICLE Here we use hydrogen/deuterium exchange coupled to mass potent counterparts can only recognize more open forms of the spectrometry (HDX-MS) to probe the effects of binding of several trimer. classes of bNAbs to soluble, native-like Env trimers (SOSIP.664 trimers) . HDX-MS measures the kinetics of deuterium exchange at protein backbone amides. After deuterium Results exchange under native conditions, the exchange is slowed by CD4bs-directed antibodies. A total of 103 and 120 unique acidification (pH 2.5) and low temperature (near 0 C). The peptides derived from SOSIP.664 trimers could be observed by protein is subsequently rapidly digested by an acid-active HDX, resulting in net sequence coverages of 92% and 82% for protease, pepsin, and the resulting peptides are resolved by isolates KNH1144 and BG505, respectively (Supplementary liquid chromatography–MS to track the kinetics of deuterium Figs 1,2). To study the interactions of NAbs with the CD4bs, exchange throughout the protein. Amides that are engaged in we compared the HDX profiles of unliganded trimers with their stable hydrogen bonds through secondary structure or that are complexes with Fabs derived from the highly potent bNAbs heavily solvent occluded are protected from exchange, resulting in VRC01 and PGV04 (IC values of 0.32 and 0.14mgml , slow deuterium uptake while amides that are in less structured respectively) and the less potent bNAb b12 (IC , 1 39 regions exhibit rapid deuterium uptake . HDX-MS is therefore a 2.82 mgml ) . The SOSIP.664 trimers were incubated with a sensitive probe of local structural dynamics under native threefold molar excess of each ligand (Fab:monomer) to favour conditions and a particularly powerful tool for monitoring full occupancy (Supplementary Fig. 3). VRC01 exerted only local structural changes resulting from ligand binding. We have effects on KNH1144 SOSIP.664 trimers, the changes being previously used HDX-MS to probe the solution behaviour of limited to increased protection of the CD4 binding loop, V5, SOSIP.664 trimers and the effects of CD4 binding . Here we now Loop D and the a1 helix in layer 2 (Fig. 2a). Nearly identical data examine the effects of a diverse panel of NAbs against several were obtained for VRC01 and the BG505 SOSIP.664 trimers epitope clusters that span a range of potencies and breadth (Supplementary Fig. 4a). All the above changes are localized to (Fig. 1b,c). The comparisons reveal that the highly potent bNAbs regions of gp120 that are in direct contact with the antibody, as recognize the pre-fusion, closed form of Env, while their less judged from the gp120-Fab structure (Fig. 2c). Antibody KNH1144 ± VRC01 Fab V2 V1 V2 V3 V4 V5 FP HR1 HR2 V1 CD4-binding loop V3 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h V5 Loop D Loop D CD4 loop V5 Layer 2 +VRC01 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number KNH1144 ± b12 Fab V1 V2 V3 V4 V5 FP HR1 HR2 176–179 V2 V1 CD4-binding loop V3 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h Bridging sheet CD4 loop V1/V2 Layer 2 Layer 2 FPPR V3 +b12 50 100 150 200 250 300 350 400 450 500 550 600 650 FPPR Residue number Figure 2 | Effects of binding of CD4bs-targeting bNAbs. Butterfly plots show the exchange profiles of SOSIP.664 trimers either unliganded (positive y axis) or as complexes with (a) VRC01 Fab or (b) b12 Fab (negative y axis). The per cent exchange for each observable peptide is plotted at the position of the centre of the peptide on the primary sequence for each time point (3 s to 20 h). The difference plots below each primary plot reveal regions undergoing slower exchange (more protected, above the zero) and faster exchange (less protected, below the zero). Individual plots for the peptides at V1/ V2 and V3 are shown in Fig. 6, and plots for every peptide are in Supplementary Figs 1 and 2. (c,d) Differences are mapped onto the closed, pre-fusion trimer crystal structure (PDB accession code: 4NCO) , with regions of faster (red) or slower exchange (blue) in the presence of each antibody highlighted. Segments unresolved in the crystal structure (V2, V4 and FPPR) are shown as dashed lines. As a reference, the binding surfaces of each bNAb on gp120 21,22 9,17,18 are shown in green (PDB: 3NGB, 2NY7) . Although b12 binds a more open conformation of the trimer , for the sake of comparison all data are illustrated on the closed, pre-fusion structure. NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 3 & 2015 Macmillan Publishers Limited. All rights reserved. Δ% Exchange % Exchange Δ% Exchange % Exchange +b12 Free +VRC01 Free No data More protected No change Less protected ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 PGV04, which binds the same surface as VRC01 but is even more deuterium labelling, here the association time with the ligand is 8,11 potent , caused virtually identical, localized increases in varied, followed by a constant 3-s deuterium pulse. The protection (Supplementary Fig. 4b). KNH1144 trimers had bound 17b Fab uniformly after 72 h, As b12 binds poorly to the BG505 SOSIP.664 trimer and fails while no such binding was detectable for their BG505 counter- to neutralize the corresponding virus , only the KNH1144 parts (Supplementary Fig. 6a,b). Both BN-PAGE and HDX SOSIP.664 trimer was used to assess what changes resulted from show that the entire population of KNH1144 SOSIP.664 trimers its binding. Unlike with VRC01 and PGV04, changes throughout was present as a 17b complex, and not just a subpopulation. the trimer were observed with b12 bound. The changes included Conversely, the BG505 SOSIP.664 sample contained no decreased protection for the majority of V1/V2, V3, layer 2 of the subpopulation that was able to bind 17b. SDS–PAGE gp120 inner domain and even within the FPPR in gp41, analysis showed no signs of trimer degradation during the long indicating that these regions become more disordered (Fig. 2b). incubation times used for the experiments above (Supplementary Only for the b21 strand of the bridging sheet, residues 176–179 in Figs 3 and 6d). V2 (sequence FYRL) and the CD4 binding loop (at the early time The rate of 17b binding to unliganded KNH1144 SOSIP.664 points) were any increases in protection observed. Based on the trimers was also assessed by immunoprecipitation, which showed b12-Fab structure with core gp120, several of the perturbed that 17b bound slowly, that is, on a timescale of hours to days regions are not proximal to the binding interface, implying that (Fig. 4c). As additional controls, we also tested the rate of 17b b12 binding is associated with longer-range structural changes in binding in the presence of sCD4 and NBD-556 (which both the trimer (Fig. 2d). induce the formation of the 17b epitope), and BMS-806 (which 19,43 inhibits 17b binding) . In the presence of NBD-556 or sCD4, 17b bound efficiently within 1 h, whereas with BMS-806 there was Glycan-dependent antibodies. When the binding of the glycan- no 17b binding even after 72 h. dependent antibodies PG9, PGT123 and 2G12 was similarly In light of the slow binding of 17b, we wondered whether the probed using HDX, we observed only minimal effects on the CD4bs antibody b12 could react better with BG505 trimers trimer. As the KNH1144 trimers lack the critical glycan at N160 if the incubation period was extended. As noted above, b12 required for PG9 binding, this complex could only be examined binds poorly to BG505 SOSIP.664 trimers, but interacts readily using BG505 trimers. The binding of PG9 resulted in increased with BG505 gp120 and non-SOSIP gp140, indicating that all protection at only a single site, residues 166–175 in V2 (Fig. 3a,c). necessary contact residues for b12 are present in the BG505 These residues are known to be proximal to the PG9 epitope, sequence . BN–PAGE analysis showed some evidence of which is centred on the N160 glycan in the crystal structure increased smearing of BG505 SOSIP.664 trimers over time, between PG9 and a V1/V2 scaffold . The mass spectra for the possibly indicating a low level of b12 complex formation (Fig. 4a). protected peptide in V2 displayed increased peak broadening Using the same HDX pulse-labelling strategy employed indicative of the presence of multiple V2 conformations in the with 17b, a change in the V2 region was visible with extended PG9-bound trimer (see Supplementary Fig. 5). incubations with b12 (Supplementary Fig. 6c). Qualitatively, these The binding of the PGT123 Fab to SOSIP.664 trimers also had results suggest that BG505 SOSIP.664 trimers can only minor effects on the HDX profiles. The observed changes bind b12 but do so very slowly. This observation is consistent were limited to increased protection at the centre of V3 and a with the lack of neutralizing activity seen with assays that small decrease for V2 residues 176–179 (Fig. 3b). The affected involve a short (30–60 min) incubation period between b12 regions are proximal to the expected PGT123 binding site, as and the BG505 virus assessed from the electron microscopy and X-ray crystal structures of the complex between the BG505 SOSIP.664 trimer and the closely related bNAb, PGT122 (ref. 41) (Fig. 3d). CD4i antibody binding to SOSIP.664 trimers probed by HDX. Antibody 2G12 has evolved a unique domain swap to The HDX profile of the fully formed complex between 17b and enable both Fabs arms to bind to one glycan cluster on gp120 the KNH1144 SOSIP.664 trimers showed that extensive changes (ref. 42). For this reason, 2G12 binding was examined using intact had occurred throughout the Env protein (Fig. 5a,c). Of note is IgG rather than the Fab fragment. The exchange profile showed that most of the changes are similar to those seen on sCD4 that 2G12 binding did not change the protection of the binding (Fig. 6a and Supplementary Fig. 4c), including increased underlying protein structure at any site in the trimer (Fig. 3c,f), protection throughout portions of the gp120 inner domain and even though the complex was fully formed (Supplementary HR1, and the loss in protection for V1/V2, V3 and the FPPR. Fig. 3a). However, unlike with sCD4, all four strands of the bridging sheet became more protected with 17b binding and no significant change was seen in V5. Slow binding of the CD4i antibody, 17b. Unlike the highly The sCD4 complex and the ternary complex with sCD4 potent bNAbs, the CD4i antibody 17b bound very slowly to the and 17b were compared, to assess the changes imparted by 17b to SOSIP.664 trimers. For the KNH1144 trimers, extended incuba- the CD4-bound conformation of the KNH1144 SOSIP.664 tions of up to 72 h were necessary to generate a homogeneous trimer. The addition of 17b had predominantly local effects near complex, while no binding of the 17b Fab to their BG505 its binding site, with the four strands of the bridging sheet, each counterparts could be detected after even this prolonged period becoming substantially more protected (Fig. 5b,d). A slight (Fig. 4a,b). In contrast, soluble two-domain CD4 (sCD4) bound increase in protection was also evident throughout layer 1 of the efficiently to both SOSIP.664 trimers within minutes, as did 17b inner domain and the CD4 binding loop was somewhat more to the CD4-complexed trimers. We note that blue native– protected within the ternary complex than with sCD4 alone. polyacrylamide gel electrophoresis (BN–PAGE) gels show only Finally, there was a small, but reproducible increase in protection two predominant bands rather than a ladder during the forma- for residues 642–645 of gp41 HR2. tion of the 17b complex with unliganded KNH1144 SOSIP.664 trimers, suggesting that when binding does occur it is a concerted event involving all three protomers (Fig. 4b). Discussion The kinetics of complex formation with 17b were also probed The collective comparison of the HDX profiles of SOSIP.664 by HDX using a pulse-labelling strategy. Unlike continuous trimers in complex with different classes of bNAbs reveals the 4 NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ARTICLE N160 BG505 ± PG9 Fab V2 V1 V2 V3 V4 V5 FP HR1 HR2 V1 V3 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h V2 50 100 150 200 250 300 350 400 450 500 550 600 650 +PG9 Residue number BG505 ± PGT123 Fab N301 V2 V1 V2 V3 V4 V5 FP HR1 HR2 V1 N332 V3 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h V3 V2 50 100 150 200 250 300 350 400 450 500 550 600 650 +PGT123 Residue number KNH1144 ± 2G12 IgG V1 V2 V3 V4 V5 FP HR1 HR2 50 100 150 200 250 300 350 400 450 500 550 600 650 V4 Residue number 3 s 1 min 30 min 20 h 50 100 150 200 250 300 350 400 450 500 550 600 650 +2G12 Residue number Figure 3 | Effects of binding of glycan-dependent bNAbs. Butterfly plots show the exchange profiles of SOSIP.664 trimers either unliganded or as complexes with (a) PG9 Fab, (b) PGT123 Fab and (c) 2G12 IgG. The differences are mapped onto the trimer crystal structure (d–f), as described in Fig. 2. As a reference, the approximate positions of the Fabs are shown as transparent surfaces (modelled from PDB accession codes 3NGB, 3U4E and 4NCO) , 2,28,30 while glycans known to be relevant to the epitopes are shown as magenta sticks . No structure is yet available for the PGT123 trimer complex; hence, 2,10 PGT122 is shown instead, as its epitope is very similar (PDB: 4NCO) extent of allosteric effects imparted by each interaction. With the not observe peptides that included residues 129–164 and 326–369 most potent neutralizing antibodies studied here, the changes on we could not monitor changes in those regions (Supplementary bNAb binding are limited to increased protection at the Fig. 2). Despite this limitation, the comparisons definitively show immediate contact regions (Figs 2 and 3). The highly potent that the glycan-dependent antibodies do not exert long-range CD4bs antibodies, VRC01 and PGV04, and all the glycan- effects on the protein framework of SOSIP.664 trimers. dependent antibodies had only limited, focused effects on the One peculiarity of PG9 is that it binds asymmetrically to Env HDX profiles of the trimers. The glycan cluster-targeting 2G12 trimers with a stoichiometry of 1:1 (Fab:trimer) . Such antibody had no effects at all on the underlying protein backbone, asymmetric binding would result in complex exchange kinetics, implying that this epitope does indeed involve only glycans. It as the three lobes of the trimer would not be affected equally. We remains possible that V4, which is proximal to the 2G12 epitope, observed a subtle broadening in the mass spectrum specifically at undergoes some changes, but this loop could not be monitored by V2 within the PG9-bound SOSIP.664 trimer, consistent with the HDX due to its heavy glycosylation. Similarly, PG9 and PGT123 existence of two V2 conformational subpopulations. No such binding may affect more areas of V1/V2 and V3, but as we could effect was seen with unliganded trimers or any other NAb NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 5 & 2015 Macmillan Publishers Limited. All rights reserved. 2G12 Δ% Exchange % Exchange Δ% Exchange % Exchange Δ% Exchange % Exchange +2G12 Free +PGT123 Free +PG9 Free No data More protected No change Less protected ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 (Fig. 2d). This outcome is in direct contrast to the purely local effects that were observed with the much more potent CD4bs bNAbs VRC01 and PGV04. Such an unexpected effect probably results from b12 binding to and stabilizing a trimer conformation that has less ordered secondary structure, resulting in faster amide exchange kinetics despite the decreased solvent accessi- bility arising from solvent occlusion from the protein–protein interface. The binding of b12 also yielded a subtle increase in 1,084 protection for residues 176–179 within V2 (sequence FYRL), something that was not observed with any of the other antibodies (Fig. 6). A structural alignment to the b12 Fab-gp120 core crystal structure indicated that a portion of the b12 paratope on the 480 trimer is adjacent to this region of V2 (Fig. 2d). Hence, residues 176–179, a region absent from the construct used to generate the BG505 b12-gp120 core crystal structure, may contribute directly to the b12 epitope. This notion is supported by the substantial effects that mutations involving residues 176–180 have on both b12 7,44,45 binding and neutralization . It is noteworthy that 17b can bind to KNH1144 SOSIP.664 trimers, but not their BG505 counterparts, in the absence of CD4. The CD4i epitope is not accessible on the unliganded pre-fusion 10,11 BG505 SOSIP.664 trimers . How, then, can 17b bind to the 1,084 corresponding KNH1144 trimers, albeit with very slow kinetics? As 17b binding in the presence of sCD4 or NBD-556 occurs rapidly, the observed slow binding to the closed trimers (that is, no CD4) cannot simply be explained by a slow intrinsic on-rate (k ). We propose that Env trimers, even in the absence of CD4, on undergo transient structural fluctuations to sample an open state, KNH1144 and in doing so the CD4i epitope is formed and exposed. This hypothesis would explain the slow binding kinetics of 17b to the unliganded KNH1144 SOSIP.664 trimers; specifically, 17b binds DMSO BMS-806 NBD-556 only to the transiently open state of the trimers, forming a complex primarily via conformational selection (Fig. 7a). This concept is also consistent with the coordinated binding of three 17b Fabs per trimer (that is, the lack of evidence for partial occupancy). Thus, once opened, the binding of 17b to a single lobe of the trimer blocks the re-formation of the quaternary interactions at the apex, which means that the other two lobes remain accessible for the rapid binding of two more 17b Fabs. In contrast to 17b, CD4 binds rapidly to the trimer, suggesting that major conformational transitions do not need to occur before binding. Instead, it is the CD4 binding event itself that most probably leads to large-scale conformational changes in the WB α Env trimer (Figs 4 and 7a). Figure 4 | Kinetics of 17b Fab binding to SOSIP.664 trimers. (a) BG505 The argument that 17b recognizes an open form of Env trimers and (b) KNH1144 SOSIP.664 trimers were incubated with ligands for is further supported by the HDX comparisons. All the major various times (from 3 min to 72 h) and resolved by BN–PAGE. (c) 17b was changes associated with the opening of the trimer, including loss incubated with KNH1144 SOSIP.664 trimers for various times, the of protection at V1/V2 and V3, were observed in the 17b complex complexes captured by protein G beads, resolved by SDS–PAGE and (Fig. 6). There were only two major differences between the 17b- visualized by immunoblotting. Identical experiments performed in the and sCD4-bound data sets: (1) for 17b, there was increased presence of BMS-806, NBD-556 and sCD4 reveal that NBD-556 and sCD4 protection throughout the bridging sheet, which is in direct both accelerate 17b binding, while BMS-806 blocks it altogether. The gp120 contact with the Fab and (2) for sCD4, there was more protection band derived from SOSIP.664 trimers is in the highlighted region marked by at V5, which is involved in the binding interface for sCD4 but not the arrow. The other bands represent nonspecific binding of the anti-human 17b. When 17b binds to the already opened sCD4-SOSIP.664 serum used as the secondary antibody. trimer complex, the major changes involve stabilization of the regions in direct contact with the Fab (Fig. 5d). We noted that the binding of b12 also caused changes throughout the trimer, with complex. Qualitatively, the degree of protection indicates that a portions of V1/V2 and V3 losing protection. Together with the large proportion of the V2 subdomains become more protected in slow binding of b12 Fab to the BG505 SOSIP.664 trimers, this the PG9 trimer complex. The stabilizing effect of a single bound finding suggests that similar to 17b, b12 recognizes an open PG9 Fab therefore probably extends beyond a single lobe of the conformation of the trimer. trimer, which is consistent with our earlier proposal that the The current study demonstrates that highly potent bNAbs binding interface involves two protomers of the trimer . target the closed conformation of Env without inducing The less potent CD4bs bNAb b12 had a unique effect in that it conformational changes, something that has been observed for resulted in changes throughout the KNH1144 SOSIP.664 trimer, several other Ab–pathogen systems . Less-potent antibodies (for with a loss of protection within some of the contact surface example, b12) may require the trimer to transiently sample 6 NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Unliganded +sCD4 (3 m) +sCD4 & 17b (3 m) +17b (3 m) +17b (4 h) +17b (72 h) +b12 (3 m) +b12 (4 h) +sCD4 (3 m) +b12 (72 h) +17b (3 m) +17b (3 m) +17b (10 m) +17b (4 h) +17b (72 h) (+) Unliganded NC 1 h 12 h 72 h 1 h 12 h 72 h 1 h 12 h 72 h 1 h sCD4 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ARTICLE KNH1144 ± 17b Fab V1 V2 V3 V4 V5 FP HR1 HR2 V2 V1 CD4-binding loop V3 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h Bridging sheet Layer 2 Layer 3 CD4 loop * HR1 Layer 3 Layer 1 FPPR V3 V1/V2 50 100 150 200 250 300 350 400 450 500 550 600 650 +17b HR1 FPPR Residue number KNH1144-sCD4 ± 17b Fab V1 V2 V3 V4 V5 FP HR1 HR2 V3 CD4-binding 0 loop V1/V2 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h Bridging sheet CD4 loop HR2 Bridging Layer 1 sheet +17b and sCD4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number Figure 5 | Effects of CD4i antibody binding. The butterfly and difference plots compare unliganded SOSIP.664 trimers (isolate KNH1144) with (a) the binary complex with 17b Fab and (b) the ternary complex with sCD4 and 17b Fab. (c) The differences are mapped onto the BG505 SOSIP.664 trimer crystal structure (PDB accession code: 4NCO), as described in Fig. 2. Contacts with CD4 (yellow) and Fabs (green) are shown as transparent surfaces (modelled relative to the gp120 core from PDB: 1GC1) . The regions unresolved in the trimer structure are shown as dashed lines. (d) The effects of 17b binding to sCD4-bound SOSIP.664 trimers are similarly mapped onto the CD4-bound conformation of gp120, as modelled into the trimer structure (PDB: 3JWD) . The change within HR2 in the ternary complex is not shown, as this region is not resolved in the 4NCO trimer structure. 100 100 100 100 50 50 50 50 (V1) (V2) (V3) (V2) (149–159) (164–175) (288–317) (166–175) KEMEGEIKNCSF EIRDKKQKVYSL LVEPVRI...VRIGPGQAF RDKKQKVYSL 0 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 110 10 10 10 10 110 10 10 10 10 110 10 10 10 10 110 10 10 10 10 Time (s) Time (s) Time (s) Time (s) 100 100 100 100 V2 50 50 50 50 (V2) (180–193) (V3) (V3) (176–179) DVVPINQGNSS... (317–324) (317–325) FYRL ...SKNSSEYRL FATGDIIG YATGDIIGD 0 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 110 10 10 10 10 110 10 10 10 10 110 10 10 10 10 110 10 10 10 10 Time (s) Time (s) Time (s) Time (s) Trimer only sCD4 b12 VRC01 PGT123 2G12 17b sCD4 and 17b PGV04 PG9 Figure 6 | Individual exchange profiles of peptides covering V1/V2 and V3. The plots for selected peptides derived from (a) KNH1144 and (b) BG505 SOSIP.664 trimers in unliganded trimer or in complex with sCD4 or various antibodies, as stated. Dots show the mean uptake values at each time point with error bars showing the s.d. between duplicate measurements (in many cases, the error bars are too small to see). Plots for all observable peptides are shown in Supplementary Figs 1 and 2. NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 7 & 2015 Macmillan Publishers Limited. All rights reserved. % Exchanged % Exchanged Δ% Exchange Δ% Exchange % Exchange % Exchange % Exchanged % Exchanged % Exchanged % Exchanged +17b +17b Free +sCD4 and sCD4 % Exchanged % Exchanged No data More protected No change Less protected A ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 10–12,19,47 bridging sheet . Moreover, quaternary steric hindrance effects restrict how b12 can gain access to the CD4 binding site on 9,17 the closed, pre-fusion trimer , a constraint that is absent from Env Env closed open monomeric gp120. The highly ordered, unliganded conformation of the Env 17b CD4 trimer in which the trimer apex is formed by the V1/V2 and V3 loops, and in which conserved epitopes such as the co-receptor Env ·CD4 Env ·17b closed open binding site are masked, is often referred to as the ‘closed’ conformation or ‘ground state’ . The binding of CD4 induces profound allosteric changes such that the trimer adopts an ‘open’ Env ·CD4 configuration, with the quaternary associations between V1/V2 open and V3 at the trimer apex now released. Cryo-electron tomography images of the unliganded (closed) and CD4-bound (fully open) trimer provide a low-resolution, but highly Tier 2+ Tier 1 CD4 independent 9,17,18 informative, view of these two states . We note that a much earlier usage of the ‘open/closed’ terminology refers to the conformations of Env on viruses, as inferred from their neutralization phenotypes. In the 1990s, it was common to refer to neutralization-sensitive (usually cell line adapted) viruses as ‘Closed’ ‘Open’ having a more open (that is, antibody accessible) form of Env gp120 gp41 V1/V2 V3 49–52 than their resistant, primary isolate counterparts . The phenotypic difference between the neutralization-sensitive, cell Figure 7 | Conformational sampling of unliganded Env trimers. (a) CD4 line-adapted, tier 1 isolates and more resistant primary isolates can bind the closed form of Env and allosterically induce an open (now categorized as tier 2 or 3) has been attributed to undefined conformation. In contrast, 17b can only bind to unliganded Env when the 7,26,53,54 aspects of the quaternary structure of their trimers . open conformation is transiently sampled, and by doing so it stabilizes this A very recent paper emphasizes the critical role of the V2 loop in conformation. (b) Env trimers exist predominantly in the closed form in these neutralization phenotypes, probably via influences on the which the interactions between V1/V2 from each protomer are intact and formation and stability of the inter-protomer associations at the V3 is occluded. Transient sampling of open conformations disrupts the trimer apex . inter-protomer interactions at the trimer apex. The trimers on higher tier Here we propose that the subtly differing open/closed (more neutralization-resistant) viruses are less likely to sample open terminologies may be manifestations of a single phenomenon: conformations, thereby protecting conserved epitopes from NAbs. Tier 1 the transient opening motions of the trimer that occur at different primary and cell line-adapted isolates sample the open conformation more rates among different isolates that are a major determinant often or for longer periods, which renders these viruses more vulnerable to of neutralization susceptibility (Fig. 7b). Under the immune neutralization. CD4-independent isolates (which are extremely pressures that target conserved regions such as the co-receptor neutralization sensitive) have evolved mutations that stabilize the open binding site, the more neutralization-resistant isolates have state, probably to facilitate co-receptor binding when CD4 is either present evolved Env trimers that better mask conserved epitopes by at low levels or entirely absent. sampling the open conformations only transiently and infre- 48–50,56,57 quently . Conversely, cell line-adapted isolates that were cultured in the absence of antibody selection pressures evolved to partially open states in which their epitopes are exposed. sample the open state more readily. CD4-independent isolates, Antibodies such as 17b whose epitopes are masked in the which can enter cells that lack CD4 but express the co-receptor, 58,59 closed trimer conformation are likely to recognize only the fully are extremely susceptible to neutralization and primarily exist open trimer conformation in which the co-receptor binding site is in the open state . This trend is consistent with the recent unmasked and its elements assembled. This trend is consistent observation that a higher level of ‘intrinsic reactivity’ (ability of with low resolution cryo-electron tomography studies, which Env to sample different conformations) correlates with sensitivity reported that sCD4 and 17b binding resulted in open trimer to antibody neutralization . Our study is limited to the conformations, that b12 binding caused the trimer to partially KNH1144 and BG505 SOSIP.664 constructs, which represent 9,17,18 open, and that VRC01 binding caused no such opening . the only soluble native-like Env trimers currently available. Furthermore, both CD4 and b12 have been observed to induce Examination of native-like trimers from other isolates will be gp120 shedding from virus, a presumed marker of substantial necessary for a complete understanding of the relationship conformational changes, whereas VRC01 and PGV04 do not have between the kinetics of Env’s transient opening motions and 45,46 such an effect . neutralization susceptibility. It is relevant to draw a distinction between what happens when Although our HDX analysis has revealed much about the NAbs bind to the gp120 subunits of trimers compared with conformational state of the trimer, under the current conditions it individual gp120 monomers. For example, VRC01 binding to does not appear to provide a quantitative measure of the kinetics gp120 monomers is associated with a large entropic penalty, of transient opening motions. If the open conformations persisted suggesting that gp120 transitions from a highly dynamic in solution on a long timescale (seconds), then it would result in form to a more constrained conformation . In contrast, b12 correlated exchange kinetics (EX1 regime) as evidenced by binding to monomeric gp120 is associated with a much less bimodal mass spectra under our experimental conditions . significant change in entropy, suggestive of a less profound However, no such EX1 behaviour was observed for the structural change . This pattern is the inverse of what we see unliganded trimer and all portions of the protein exchange when the same two NAbs interact with the trimer; the probable through fast local structural fluctuations (EX2). Furthermore, explanation is that gp120 in the trimer context is more ligands that are expected to stabilize the closed conformation (for conformationally constrained then when it is an isolated example, PG9) only show local effects at their binding site, monomer, particularly with respect to V1/V2, V3 and the without altering the HDX profile through the rest of the trimer 8 NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ARTICLE apex. Likewise, under the experimental conditions used, Hydrogen/deuterium exchange. SOSIP.664–antibody complexes (15 mg) were diluted tenfold into deuterated PBS buffer and incubated at room temperature. SOSIP.664 from BG505 and KNH1144 isolates had very similar Aliquots removed after 3 s, 1 min, 30 min and 20 h were quenched by mixing with HDX profiles, despite phenotypic differences. For one, KNH1144 an equal volume of cold 200 mM Tris-2-carboxyethyl phosphine, 0.2% formic acid 19,32 has lower thermal stability than BG505 trimers . The kinetics (final pH 2.5). The samples were subsequently digested with pepsin (at of b12 and 17b binding are also drastically different between the 0.15 mg ml ) for 5 min on ice, flash frozen in liquid nitrogen and stored at 80 C. For liquid chromatography–MS analysis, samples were thawed on ice for two isolates, indicating a lower frequency of exposure of several 5 min and manually injected onto a Waters BEH 1.7 mm, 1.2 5 mm trap column epitopes in BG505. The relative kinetics of ligand binding to the (Waters) flowing 0.1% TFA at 200mlmin . After 3 min of washing, the peptides partially open (with b12) or fully open (with 17b) form of the were resolved over a Hypersil 1 50 mm, 2.1 mm C18 column (Thermo Scientific), trimer should serve as an additional assay for assessing the degree using a gradient of 15%–40% B in 8 min (A: 0.05% TFA, 5% ACN; B: 0.05% TFA, 80% ACN). Two hundred microlitres of 10% formic acid, 80% MeOH, 2:1 of transient opening motions within different Env trimers. IPA:ACN and 80% ACN were injected over the loop and trap column between Recently, single-molecule fluorescence experiments have injections, while two rapid (10%–100% B in 30 s) gradients were used to clean the demonstrated that native Env on the membrane samples transient resolving column, to minimize carryover. Eluting peptides were analysed with a open states, and that potent neutralizing antibodies stabilize the Waters Synapt Q-TOF mass spectrometer, with source and desolvation tempera- 12,48 closed state of the trimer . Furthermore, in agreement with tures of 80C and 150 C, respectively. Peptic peptides were identified by exact mass and MS/MS spectra. Identification our own observations, the transient fluctuations to the activated of glycopeptides was aided by MS/MS spectra of the fully deglycosylated samples as (open) states were more frequent with neutralization-sensitive described previously . Per cent exchange for each time point was calculated isolates. Despite any caveats that may be associated with the use relative to zero and fully deuterated standards as described previously . For ligand of soluble SOSIP.664 trimers (that is, the presence of stabilizing binding kinetics, SOSIP.664 trimers at 1 mg ml were incubated with a threefold excess of b12 or 17b Fab in PBS at 22 C. At 3 min, 4 h and 72 h, an aliquot of the mutations and the absence of the MPER/transmembrane mixture was diluted tenfold into deuterated PBS buffer for 3 s and rapidly segments of gp41), their properties and behaviour do appear to quenched. reflect what is observed using full-length, fusion-competent Env trimers. Beyond the concurrence in behaviours between soluble SOSIP.664 trimers and Env on virus, characterizing the Antibody binding assays. The kinetics of 17b binding were measured by incu- interaction of the SOSIP trimer with antibodies provides bating SOSIP.664 trimers at 1 mg ml with a threefold molar excess of 17b Fab (relative to protomers) and a monitoring complex formation by BN–PAGE. valuable information for understanding its properties as a Immunoprecipitation experiments were initiated by addition of 2 mg of 17b Fab to potential immunogen. 3 mg of KNH1144 SOSIP.664 trimers in 120ml of PBS plus 2% dimethyl sulfoxide, The development of broadly cross-reactive antibodies is together with the following: no ligand, 100mM NBD-556, 100mM BMS-806 or relatively uncommon during the course of HIV-1 infection and 0.4 mM of sCD4. Pre-washed protein G sepharose beads were added after the mixtures were incubated at 25 C for various times, and rotated on a nutator for generally takes several years when it does happen . In light 1 h. The beads were pelleted by centrifugation and washed four times in PBS plus of the fact that all of the highly potent bNAbs target the closed 0.1% Tween 20, followed by re-suspension and boiling in reducing SDS-gel loading form of Env, more potent Ab responses may be achieved by buffer (Invitrogen). Western blottings were probed with HIV-Ig serum (1:1,000 using trimer immunogens that present the closed form of dilution), followed by anti-human-horseradish peroxidase (1:5,000 dilution; Jack- son Immunoresearch). The labelled bands were visualized by chemilluminescence Env as stably as possible. For such immunogens, any transient (Western lightning, Perkin Elmer). sampling of open conformations should be minimized to avoid exposing undesirable, non-neutralizing epitopes that could potentially distract the humoral immune response. The BG505 References SOSIP.664 protein appears to embody such an immunogen 1. Walker, L. M. & Burton, D. R. Rational antibody-based HIV-1 vaccine design: that presents the major neutralizing epitopes in the context current approaches and future directions. Curr. Opin. Immunol. 22, 358–366 of a stable, closed trimer . The contrast between the KNH1144 (2010). 2. Walker, L. M. et al. Broad neutralization coverage of HIV by multiple highly and BG505 SOSIP.664 trimers, which bind 17b slowly versus not potent antibodies. Nature 477, 466–470 (2011). at all, implies that the BG505 trimers are the more likely to 3. Mascola, J. R. & Haynes, B. F. HIV-1 neutralizing antibodies: understanding remain in the fully closed state, sampling the open state(s) less nature’s pathways. Immunol. Rev. 254, 225–244 (2013). frequently than their KNH1144 counterparts. Further 4. Wyatt, R. et al. The antigenic structure of the HIV gp120 envelope modifications to the BG505 SOSIP.664 trimers may be useful glycoprotein. Nature 393, 705–711 (1998). 5. Kwong, P. D. et al. HIV-1 evades antibody-mediated neutralization through for reducing the spontaneous or CD4-induced opening of the conformational masking of receptor-binding sites. Nature 420, 678–682 (2002). trimer that is likely to occur in vivo during the course of 6. Wei, X. et al. Antibody neutralization and escape by HIV-1. Nature 422, vaccination. Identifying other closed conformation, native-like 307–312 (2003). trimers with comparable properties is an additional priority, not 7. Pantophlet, R. et al. Fine mapping of the interaction of neutralizing and least to allow the exploration of more complex immunization nonneutralizing monoclonal antibodies with the CD4 binding site of human regimens. immunodeficiency virus type 1 gp120. J. Virol. 77, 642–658 (2003). 8. Wu, X. et al. Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing. Science 333, 1593–1602 (2011). 9. Liu, J., Bartesaghi, A., Borgnia, M. J., Sapiro, G. & Subramaniam, S. Molecular Methods architecture of native HIV-1 gp120 trimers. Nature 455, 109–113 (2008). Sample preparation. SOSIP.664 trimers based on the subtype A isolates 10. Julien, J. P. et al. Crystal structure of a soluble cleaved HIV-1 envelope trimer. KNH1144 and BG505 were expressed in 293S GnTI / cells and purified by Science 342, 1477–1483 (2013). 2G12 affinity followed by size-exclusion chromatography (Superdex S200, GE 11. Lyumkis, D. et al. Cryo-EM structure of a fully glycosylated soluble cleaved Healthcare) in PBS (20 mM sodium phosphate pH 7.4, 150 mM NaCl with 1 mM HIV-1 envelope trimer. Science 342, 1484–1490 (2013). 19,63 EDTA and 0.02% sodium azide) as described previously . sCD4 (ref. 64), HIV- 12. Pancera, M. et al. Structure and immune recognition of trimeric pre-fusion 4,14,23,24 IG and monoclonal antibodies 17b , b12 (refs 20,65,66), 2G12 (refs 27,67) HIV-1 Env. Nature 514, 455–461 (2014). and VRC01 (ref. 68) were obtained from the NIH AIDS reagents programme. Fabs 13. Wu, L. et al. CD4-induced interaction of primary HIV-1 gp120 glycoproteins were prepared using Pierce Fab preparation kit (Thermo Scientific) according to with the chemokine receptor CCR-5. Nature 384, 179–183 (1996). manufacturer’s instructions. PGV04, PGT123 and PG9 Fabs were prepared as 69 14. Trkola, A. et al. CD4-dependent, antibody-sensitive interactions between HIV- described previously . All proteins were buffer exchanged into PBS. Complexes 1 and its co-receptor CCR-5. Nature 384, 184–187 (1996). with SOSIP.664 trimers were formed by overnight incubation with a threefold 15. Rizzuto, C. D. et al. A conserved HIV gp120 glycoprotein structure involved in molar excess of ligand (relative to each protomer) at 4 C, except for the 17b Fab chemokine receptor binding. Science 280, 1949–1953 (1998). complex, which was incubated for 72 h at room temperature. SDS– and BN–PAGE analyses were performed with the same samples used for HDX, to ensure that 16. White, T. A. et al. Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary complexes had formed and that no degradation had occurred throughout the incubations (Supplementary Fig. 3). structure. PLoS Pathog. 6, e1001249 (2010). NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 9 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 17. Tran, E. E. et al. Structural mechanism of trimeric HIV-1 envelope glycoprotein 47. Guttman, M., Kahn, M., Garcia, N. K., Hu, S. L. & Lee, K. K. Solution structure, activation. PLoS Pathog. 8, e1002797 (2012). conformational dynamics, and CD4-induced activation in full-length, 18. Harris, A. et al. Trimeric HIV-1 glycoprotein gp140 immunogens and native glycosylated, monomeric HIV gp120. J. Virol. 86, 8750–8764 (2012). HIV-1 envelope glycoproteins display the same closed and open quaternary 48. Munro, J. B. et al. Conformational dynamics of single HIV-1 envelope trimers molecular architectures. Proc. Natl Acad. Sci. USA 108, 11440–11445 (2011). on the surface of native virions. Science 346, 759–763 (2014). 19. Guttman, M. et al. CD4-induced activation in a soluble HIV-1 Env trimer. 49. Moore, J. P., Trkola, A., Sattentau, Q. J., Cao, Y. & Ho, D. D. Studies on the Structure 22, 974–984 (2014). interactions of monoclonal antibodies with HIV-1 isolates from clades A-F. 20. Burton, D. R. et al. Efficient neutralization of primary isolates of HIV-1 by a Proceedings of IX Colloque des Cent Gardes 151–155 (1994). recombinant human monoclonal antibody. Science 266, 1024–1027 (1994). 50. Moore, J. P., Sattentau, Q. J., Wyatt, R. & Sodroski, J. Probing the structure of 21. Zhou, T. et al. Structural definition of a conserved neutralization epitope on the human immunodeficiency virus surface glycoprotein gp120 with a panel of HIV-1 gp120. Nature 445, 732–737 (2007). monoclonal antibodies. J. Virol. 68, 469–484 (1994). 22. Zhou, T. et al. Structural basis for broad and potent neutralization of HIV-1 by 51. Sullivan, N., Sun, Y., Li, J., Hofmann, W. & Sodroski, J. Replicative function and antibody VRC01. Science 329, 811–817 (2010). neutralization sensitivity of envelope glycoproteins from primary and T-cell 23. Thali, M. et al. Characterization of conserved human immunodeficiency line-passaged human immunodeficiency virus type 1 isolates. J. Virol. 69, virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. 4413–4422 (1995). J. Virol. 67, 3978–3988 (1993). 52. Sattentau, Q. J. & Moore, J. P. Human immunodeficiency virus type 1 24. Kwong, P. D. et al. Structure of an HIV gp120 envelope glycoprotein in neutralization is determined by epitope exposure on the gp120 oligomer. J. Exp. complex with the CD4 receptor and a neutralizing human antibody. Nature Med. 182, 185–196 (1995). 393, 648–659 (1998). 53. Fouts, T. R., Binley, J. M., Trkola, A., Robinson, J. E. & Moore, J. P. 25. Salzwedel, K., Smith, E. D., Dey, B. & Berger, E. A. Sequential CD4-coreceptor Neutralization of the human immunodeficiency virus type 1 primary isolate JR- interactions in human immunodeficiency virus type 1 Env function: soluble FL by human monoclonal antibodies correlates with antibody binding to the CD4 activates Env for coreceptor-dependent fusion and reveals blocking oligomeric form of the envelope glycoprotein complex. J. Virol. 71, 2779–2785 activities of antibodies against cryptic conserved epitopes on gp120. J. Virol. 74, (1997). 326–333 (2000). 54. Kwong, P. D. et al. Structures of HIV-1 gp120 envelope glycoproteins from 26. Seaman, M. S. et al. Tiered categorization of a diverse panel of HIV-1 Env laboratory-adapted and primary isolates. Structure 8, 1329–1339 (2000). pseudoviruses for assessment of neutralizing antibodies. J. Virol. 84, 1439–1452 55. Brandenberg, O. F. et al. Partial rescue of V1V2 mutant infectivity by HIV-1 (2010). cell-cell transmission supports the domain’s exceptional capacity for sequence 27. Trkola, A. et al. Human monoclonal antibody 2G12 defines a distinctive variation. Retrovirology 11, 75 (2014). neutralization epitope on the gp120 glycoprotein of human immunodeficiency 56. Kolchinsky, P., Kiprilov, E. & Sodroski, J. Increased neutralization sensitivity of virus type 1. J. Virol. 70, 1100–1108 (1996). CD4-independent human immunodeficiency virus variants. J. Virol. 75, 28. Sanders, R. W. et al. The mannose-dependent epitope for neutralizing antibody 2041–2050 (2001). 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J. Virol. 57. Kabat, D., Kozak, S. L., Wehrly, K. & Chesebro, B. Differences in CD4 76, 7293–7305 (2002). dependence for infectivity of laboratory-adapted and primary patient 29. Murin, C. D. et al. Structure of 2G12 Fab2 in complex with soluble and fully isolates of human immunodeficiency virus type 1. J. Virol. 68, 2570–2577 glycosylated HIV-1 Env by negative-stain single particle electron microscopy. (1994). J. Virol. 88, 10177–10188 (2014). 58. Hoffman, T. L. et al. Stable exposure of the coreceptor-binding site in a CD4- 30. Walker, L. M. et al. Broad and potent neutralizing antibodies from an African independent HIV-1 envelope protein. Proc. Natl Acad. Sci. USA 96, 6359–6364 donor reveal a new HIV-1 vaccine target. Science 326, 285–289 (2009). (1999). 31. McLellan, J. S. et al. Structure of HIV-1 gp120 V1/V2 domain with broadly 59. Edwards, T. G. et al. Relationships between CD4 independence, neutralization neutralizing antibody PG9. Nature 480, 336–343 (2011). sensitivity, and exposure of a CD4-induced epitope in a human 32. Julien, J. P. et al. Asymmetric recognition of the HIV-1 trimer by broadly immunodeficiency virus type 1 envelope protein. J. Virol. 75, 5230–5239 neutralizing antibody PG9. Proc. Natl Acad. Sci. USA 110, 4351–4356 (2013). (2001). 33. Bartesaghi, A., Merk, A., Borgnia, M. J., Milne, J. L. & Subramaniam, S. 60. Haim, H. et al. Contribution of intrinsic reactivity of the HIV-1 envelope Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by glycoproteins to CD4-independent infection and global inhibitor sensitivity. cryo-electron microscopy. Nat. Struct. Mol. Biol. 20, 1352–1357 (2013). PLoS Pathog. 7, e1002101 (2011). 34. Blattner, C. et al. Structural delineation of a quaternary, cleavage-dependent 61. Weis, D. D., Wales, T. E., Engen, J. R., Hotchko, M. & Ten Eyck, L. F. epitope at the gp41-gp120 interface on intact HIV-1 Env trimers. Immunity 40, Identification and characterization of EX1 kinetics in H/D exchange mass 669–680 (2014). spectrometry by peak width analysis. J. Am. Soc. Mass Spectrom. 17, 1498–1509 35. Klasse, P. J. & Sattentau, Q. J. Occupancy and mechanism in antibody-mediated (2006). neutralization of animal viruses. J. Gen. Virol. 83, 2091–2108 (2002). 62. Stamatatos, L., Morris, L., Burton, D. R. & Mascola, J. R. Neutralizing 36. Reading, S. A. & Dimmock, N. J. Neutralization of animal virus infectivity by antibodies generated during natural HIV-1 infection: good news for an HIV-1 antibody. Arch. Virol. 152, 1047–1059 (2007). vaccine? Nat. Med. 15, 866–870 (2009). 37. Sanders, R. W. et al. A next-generation cleaved, soluble HIV-1 Env 63. Iyer, S. P. et al. Purified, proteolytically mature HIV type 1 SOSIP gp140 trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly envelope trimers. AIDS Res. Hum. Retroviruses 23, 817–828 (2007). neutralizing but not non-neutralizing antibodies. PLoS Pathog. 9, e1003618 64. Garlick, R. L., Kirschner, R. J., Eckenrode, F. M., Tarpley, W. G. & (2013). Tomich, C. S. Escherichia coli expression, purification, and biological 38. Marcsisin, S. R. & Engen, J. R. Hydrogen exchange mass spectrometry: what is activity of a truncated soluble CD4. AIDS Res. Hum. Retroviruses 6, 465–479 it and what can it tell us? Anal. Bioanal. Chem. 397, 967–972 (2010). (1990). 39. Eroshkin, A. M. et al. bNAber: database of broadly neutralizing HIV antibodies. 65. Burton, D. R. et al. A large array of human monoclonal antibodies to type 1 Nucleic Acids Res. 42, D1133–D1139 (2014). human immunodeficiency virus from combinatorial libraries of asymptomatic 40. Ringe, R. P. et al. Cleavage strongly influences whether soluble HIV-1 envelope seropositive individuals. Proc. Natl Acad. Sci. USA 88, 10134–10137 glycoprotein trimers adopt a native-like conformation. Proc. Natl Acad. Sci. (1991). USA 110, 18256–18261 (2013). 66. Barbas, 3rd C. F. et al. Recombinant human Fab fragments neutralize 41. Julien, J. P. et al. Broadly neutralizing antibody PGT121 allosterically modulates human type 1 immunodeficiency virus in vitro. Proc. Natl Acad. Sci. USA 89, CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple 9339–9343 (1992). surrounding glycans. PLoS Pathog. 9, e1003342 (2013). 67. Buchacher, A. et al. Generation of human monoclonal antibodies against 42. Calarese, D. A. et al. Antibody domain exchange is an immunological solution HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for to carbohydrate cluster recognition. Science 300, 2065–2071 (2003). peripheral blood lymphocyte immortalization. AIDS Res. Hum. Retroviruses 10, 43. Schon, A. et al. Thermodynamics of binding of a low-molecular-weight CD4 359–369 (1994). mimetic to HIV-1 gp120. Biochemistry 45, 10973–10980 (2006). 68. Wu, X. et al. Rational design of envelope identifies broadly neutralizing human 44. Mo, H. et al. Human immunodeficiency virus type 1 mutants that escape monoclonal antibodies to HIV-1. Science 329, 856–861 (2010). neutralization by human monoclonal antibody IgG1b12. J. Virol. 71, 69. Pejchal, R. et al. Structure and function of broadly reactive antibody PG16 6869–6874 (1997). reveal an H3 subdomain that mediates potent neutralization of HIV-1. Proc. 45. Li, Y. et al. Mechanism of neutralization by the broadly neutralizing HIV-1 Natl Acad. Sci. USA 107, 11483–11488 (2010). monoclonal antibody VRC01. J. Virol. 85, 8954–8967 (2011). 70. Pancera, M. et al. Structure of HIV-1 gp120 with gp41-interactive region 46. Falkowska, E. et al. PGV04, an HIV-1 gp120 CD4 binding site antibody, is reveals layered envelope architecture and basis of conformational mobility. broad and potent in neutralization but does not induce conformational changes Proc. Natl Acad. Sci. USA 107, 1166–1171 (2010). characteristic of CD4. J. Virol. 86, 4394–4403 (2012). 10 NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ARTICLE Acknowledgements Author contributions The following reagents were obtained through the AIDS Research and Reference Reagent M.G. conducted the experiments. A.C., J.-P.J. and R.W.S. provided essential reagents. Program, Division of AIDS, NIAID, NIH: sCD4-183 from Pharmacia, Inc.; HIV-1 gp120 M.G. and K.K.L. analysed the data and made the figures. M.G., J.-P.J., R.W.S., I.A.W., Monoclonal Antibody (IgG1 b12) from Dr Dennis Burton and Carlos Barbas; HIV-1 gp120 J.P.M. and K.K.L. wrote the manuscript. Monoclonal Antibody (17b) from Dr James E. Robinson; HIV-1 gp120 MAb (VRC01) from Dr John Mascola; HIV-1 gp120 Monoclonal Antibody (2G12) from Additional information Dr Hermann Katinger; HIV-IG from NABI and NHLBI. This work was supported by NIH grant F32-GM097805 (M.G.), R01-GM099989 and R21-AI112389 (K.K.L.), P01- Supplementary Information accompanies this paper at http://www.nature.com/ AI82362 (J.P.M. and I.A.W.), R01-AI084817 (I.A.W.), R37-AI36082 (J.P.M.), UAB CFAR naturecommunications grant (P30-AI027767) through the CNIHR programme for new HIV investigators (K.K.L.), Competing financial interests: The authors declare no competing financial interests. as well as the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Dis- covery (CAVD) grant OPP1033102 (K.K.L.) and the International AIDS Vaccine Initiative Reprints and permission information is available online at http://npg.nature.com/ Neutralizing Antibody Consortium and Center (I.A.W.), CHAVI-ID UM1-AI100663 reprintsandpermissions/ (I.A.W.), a Vidi grant from the Netherlands Organization for Scientific Research (NWO) (R.W.S.), a Starting Investigator Grant from the European Research Council ERC-StG- How to cite this article: Guttman, M. et al. Antibody potency relates to the 2011-280829-SHEV (R.W.S.) and a Canadian Institutes of Health Research fellowship (J.- ability to recognize the closed, pre-fusion form of HIV Env. Nat. Commun. 6:6144 P.J.). We thank Andrew Ward and Per Johan Klasse for helpful comments and suggestions. doi: 10.1038/ncomms7144 (2015). NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 11 & 2015 Macmillan Publishers Limited. All rights reserved. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Communications Springer Journals

Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env

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ARTICLE Received 11 Aug 2014 | Accepted 15 Dec 2014 | Published 5 Feb 2015 DOI: 10.1038/ncomms7144 Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env 1 2 3 2,4 3 Miklos Guttman , Albert Cupo , Jean-Philippe Julien , Rogier W. Sanders , Ian A. Wilson , 2 1 John P. Moore & Kelly K. Lee 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. 1 2 Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195, USA. Weill Cornell Medical College, New York, New York 10021, USA. Department of Integrative Structural and Computational Biology, International AIDS Vaccine Initiative Neutralizing Antibody Center, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA. Department of Medical Microbiology, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands. Correspondence and requests for materials should be addressed to K.K.L. (email: [email protected]). NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ighly potent, broadly neutralizing antibodies (bNAbs) are (CD4bs), its specific binding footprint is not identical to that of 8,21 now being used to guide the design of an effective HIV-1 CD4, which restricts its ability to target the native trimer . 1–3 Hvaccine . One major obstacle in eliciting a protective, More recently identified bNAbs, such as VRC01 (ref. 22) and broad humoral immune response has been the high antigenic PGV04 (ref. 8), more precisely target the CD4bs and have diversity among circulating isolates. The hypervariable loops significantly greater breadth and potency. present on the surface of the envelope glycoprotein (Env) trimer CD4 binding leads to large-scale conformational changes induce antibodies that are highly isolate-specific and generally within Env that create a CD4-induced (CD4i) epitope on gp120, lack breadth (as defined by the percent of HIV-1 isolates the target of some of the earliest known NAbs . One such they can neutralize). The more conserved regions of Env antibody, 17b, recognizes a portion of the bridging sheet that are masked within the trimer before its activation, or are becomes exposed after CD4 binding; its epitope overlaps with the shielded by dense glycosylation; both of these factors restrict the conserved co-receptor binding site . Although their epitopes are 4–7 generation of broadly active responses . Despite these viral conserved, CD4i antibodies such as 17b generally neutralize defenses, a subset of HIV-1-infected individuals can, over time, only lab-adapted or abnormally sensitive primary viruses develop potent (as defined by their IC values in neutralization (‘tier 1’ isolates) on which the co-receptor binding site is 25,26 assays) bNAbs that, in some cases, neutralize 490% of relatively exposed . 2,8 circulating strains . Env glycans are also now understood to be wholly or partially Structural studies have revealed much about the overall involved in the epitopes for several, particularly potent bNAbs. architecture of the Env trimer in its closed, pre-fusion form, Antibody 2G12 recognizes a conserved patch of high-mannose including the arrangement of the variable loops at the trimer apex glycans on the periphery of the CD4bs and has moderate 9–12 27–29 (Fig. 1a) . In this conformation, the V3 region is sequestered neutralization potency and breadth . The more potent PG9/ under the V1/V2 loop structure, while elements of the bridging PG16 bNAbs target the V1/V2 structure at the trimer apex via a sheet are involved in quaternary contacts below the V1/V2 loops; glycan at N160 (refs 30,31). Several bNAbs such as PGT123 that together, these interdomain interactions lead to the occlusion of interact with the base of V3, including glycans at N301 and N332, the conserved V3 and bridging sheet elements that are are among the most potent yet identified . 13–15 components of the co-receptor binding site . Binding of the The different orientations in which many of these antibodies primary receptor, CD4, induces major structural changes within bind various native-like forms of the Env trimer have been Env, including the dissociation of contacts between V1/V2 and described at low to intermediate resolution, via electron 11,17,18,32–34 V3 at the trimer apex and the coalescence of the separate microscopy . However, critical details of how they 4,16–19 elements of the co-receptor binding site . bind the trimer and, by extension, neutralize the virus remain to Defining how antibodies recognize their epitopes on simple or be understood. Although antibody binding is often sufficient to more complex forms of Env has also shed light on how the trimer impede the interaction of a trimer with CD4 or the co-receptor, functions, and how it resists neutralization (Fig. 1b). Antibody other neutralization mechanisms may involve antibodies locking b12 was among the first to be generally accepted as broadly the trimer in the closed, pre-fusion state or, alternatively, driving 20 35,36 neutralizing . Although it recognizes the CD4 binding site its destabilization and inactivation . PG9 17b 2G12 PGT123 V1/V2 CD4, b12, VRC01, V4 PGV04 V5 VRC01 PGV04 b12 75 PG9 Inner PGT123 domain HR2 (gp41) 2G12 17b Outer domain V3 Bridging sheet HR1 (gp41) Layer 1 Layer 2 Layer 3 β-Sandwich domain 0.01 0.1 1 10 Potency –1 median IC (μg ml ) Figure 1 | Neutralizing antibody targets on Env trimers. (a) The structural elements of gp120 are coloured on one protomer of the closed, pre-fusion Env trimer (PDB accession code: 4NCO) .(b) The epitopes of the various NAbs used in this study are highlighted on the structure of the closed, pre-fusion 8,21,22 trimer. The CD4bs, the target of antibodies b12, VRC01 and PGV04 is shown in orange . Glycans at N301 and N332 along the base of V3 make 41 31 up the PGT123 epitope (magenta) . The glycan at N160 and nearby residues in V1/V2 constitute the PG9 epitope (cyan) . Antibody 2G12 recognizes the cluster of glycans at positions N295, N332, N392, N386 and N448 (green) . Antibody 17b sees the CD4i epitope that is only formed and exposed after CD4 binding . The viral membrane is shown at the base to illustrate the orientation of Env (electron microscopy databank (EMD) accession code: 9  1 5022) .(c) The breadth (by per cent of viruses neutralized at concentrations o50mgml ) and neutralization potency (median IC values of the viruses neutralized) are plotted for each NAb. Neutralization data were obtained from BNAber.org . 2 NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Breadth % Viruses neutralized NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ARTICLE Here we use hydrogen/deuterium exchange coupled to mass potent counterparts can only recognize more open forms of the spectrometry (HDX-MS) to probe the effects of binding of several trimer. classes of bNAbs to soluble, native-like Env trimers (SOSIP.664 trimers) . HDX-MS measures the kinetics of deuterium exchange at protein backbone amides. After deuterium Results exchange under native conditions, the exchange is slowed by CD4bs-directed antibodies. A total of 103 and 120 unique acidification (pH 2.5) and low temperature (near 0 C). The peptides derived from SOSIP.664 trimers could be observed by protein is subsequently rapidly digested by an acid-active HDX, resulting in net sequence coverages of 92% and 82% for protease, pepsin, and the resulting peptides are resolved by isolates KNH1144 and BG505, respectively (Supplementary liquid chromatography–MS to track the kinetics of deuterium Figs 1,2). To study the interactions of NAbs with the CD4bs, exchange throughout the protein. Amides that are engaged in we compared the HDX profiles of unliganded trimers with their stable hydrogen bonds through secondary structure or that are complexes with Fabs derived from the highly potent bNAbs heavily solvent occluded are protected from exchange, resulting in VRC01 and PGV04 (IC values of 0.32 and 0.14mgml , slow deuterium uptake while amides that are in less structured respectively) and the less potent bNAb b12 (IC , 1 39 regions exhibit rapid deuterium uptake . HDX-MS is therefore a 2.82 mgml ) . The SOSIP.664 trimers were incubated with a sensitive probe of local structural dynamics under native threefold molar excess of each ligand (Fab:monomer) to favour conditions and a particularly powerful tool for monitoring full occupancy (Supplementary Fig. 3). VRC01 exerted only local structural changes resulting from ligand binding. We have effects on KNH1144 SOSIP.664 trimers, the changes being previously used HDX-MS to probe the solution behaviour of limited to increased protection of the CD4 binding loop, V5, SOSIP.664 trimers and the effects of CD4 binding . Here we now Loop D and the a1 helix in layer 2 (Fig. 2a). Nearly identical data examine the effects of a diverse panel of NAbs against several were obtained for VRC01 and the BG505 SOSIP.664 trimers epitope clusters that span a range of potencies and breadth (Supplementary Fig. 4a). All the above changes are localized to (Fig. 1b,c). The comparisons reveal that the highly potent bNAbs regions of gp120 that are in direct contact with the antibody, as recognize the pre-fusion, closed form of Env, while their less judged from the gp120-Fab structure (Fig. 2c). Antibody KNH1144 ± VRC01 Fab V2 V1 V2 V3 V4 V5 FP HR1 HR2 V1 CD4-binding loop V3 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h V5 Loop D Loop D CD4 loop V5 Layer 2 +VRC01 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number KNH1144 ± b12 Fab V1 V2 V3 V4 V5 FP HR1 HR2 176–179 V2 V1 CD4-binding loop V3 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h Bridging sheet CD4 loop V1/V2 Layer 2 Layer 2 FPPR V3 +b12 50 100 150 200 250 300 350 400 450 500 550 600 650 FPPR Residue number Figure 2 | Effects of binding of CD4bs-targeting bNAbs. Butterfly plots show the exchange profiles of SOSIP.664 trimers either unliganded (positive y axis) or as complexes with (a) VRC01 Fab or (b) b12 Fab (negative y axis). The per cent exchange for each observable peptide is plotted at the position of the centre of the peptide on the primary sequence for each time point (3 s to 20 h). The difference plots below each primary plot reveal regions undergoing slower exchange (more protected, above the zero) and faster exchange (less protected, below the zero). Individual plots for the peptides at V1/ V2 and V3 are shown in Fig. 6, and plots for every peptide are in Supplementary Figs 1 and 2. (c,d) Differences are mapped onto the closed, pre-fusion trimer crystal structure (PDB accession code: 4NCO) , with regions of faster (red) or slower exchange (blue) in the presence of each antibody highlighted. Segments unresolved in the crystal structure (V2, V4 and FPPR) are shown as dashed lines. As a reference, the binding surfaces of each bNAb on gp120 21,22 9,17,18 are shown in green (PDB: 3NGB, 2NY7) . Although b12 binds a more open conformation of the trimer , for the sake of comparison all data are illustrated on the closed, pre-fusion structure. NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 3 & 2015 Macmillan Publishers Limited. All rights reserved. Δ% Exchange % Exchange Δ% Exchange % Exchange +b12 Free +VRC01 Free No data More protected No change Less protected ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 PGV04, which binds the same surface as VRC01 but is even more deuterium labelling, here the association time with the ligand is 8,11 potent , caused virtually identical, localized increases in varied, followed by a constant 3-s deuterium pulse. The protection (Supplementary Fig. 4b). KNH1144 trimers had bound 17b Fab uniformly after 72 h, As b12 binds poorly to the BG505 SOSIP.664 trimer and fails while no such binding was detectable for their BG505 counter- to neutralize the corresponding virus , only the KNH1144 parts (Supplementary Fig. 6a,b). Both BN-PAGE and HDX SOSIP.664 trimer was used to assess what changes resulted from show that the entire population of KNH1144 SOSIP.664 trimers its binding. Unlike with VRC01 and PGV04, changes throughout was present as a 17b complex, and not just a subpopulation. the trimer were observed with b12 bound. The changes included Conversely, the BG505 SOSIP.664 sample contained no decreased protection for the majority of V1/V2, V3, layer 2 of the subpopulation that was able to bind 17b. SDS–PAGE gp120 inner domain and even within the FPPR in gp41, analysis showed no signs of trimer degradation during the long indicating that these regions become more disordered (Fig. 2b). incubation times used for the experiments above (Supplementary Only for the b21 strand of the bridging sheet, residues 176–179 in Figs 3 and 6d). V2 (sequence FYRL) and the CD4 binding loop (at the early time The rate of 17b binding to unliganded KNH1144 SOSIP.664 points) were any increases in protection observed. Based on the trimers was also assessed by immunoprecipitation, which showed b12-Fab structure with core gp120, several of the perturbed that 17b bound slowly, that is, on a timescale of hours to days regions are not proximal to the binding interface, implying that (Fig. 4c). As additional controls, we also tested the rate of 17b b12 binding is associated with longer-range structural changes in binding in the presence of sCD4 and NBD-556 (which both the trimer (Fig. 2d). induce the formation of the 17b epitope), and BMS-806 (which 19,43 inhibits 17b binding) . In the presence of NBD-556 or sCD4, 17b bound efficiently within 1 h, whereas with BMS-806 there was Glycan-dependent antibodies. When the binding of the glycan- no 17b binding even after 72 h. dependent antibodies PG9, PGT123 and 2G12 was similarly In light of the slow binding of 17b, we wondered whether the probed using HDX, we observed only minimal effects on the CD4bs antibody b12 could react better with BG505 trimers trimer. As the KNH1144 trimers lack the critical glycan at N160 if the incubation period was extended. As noted above, b12 required for PG9 binding, this complex could only be examined binds poorly to BG505 SOSIP.664 trimers, but interacts readily using BG505 trimers. The binding of PG9 resulted in increased with BG505 gp120 and non-SOSIP gp140, indicating that all protection at only a single site, residues 166–175 in V2 (Fig. 3a,c). necessary contact residues for b12 are present in the BG505 These residues are known to be proximal to the PG9 epitope, sequence . BN–PAGE analysis showed some evidence of which is centred on the N160 glycan in the crystal structure increased smearing of BG505 SOSIP.664 trimers over time, between PG9 and a V1/V2 scaffold . The mass spectra for the possibly indicating a low level of b12 complex formation (Fig. 4a). protected peptide in V2 displayed increased peak broadening Using the same HDX pulse-labelling strategy employed indicative of the presence of multiple V2 conformations in the with 17b, a change in the V2 region was visible with extended PG9-bound trimer (see Supplementary Fig. 5). incubations with b12 (Supplementary Fig. 6c). Qualitatively, these The binding of the PGT123 Fab to SOSIP.664 trimers also had results suggest that BG505 SOSIP.664 trimers can only minor effects on the HDX profiles. The observed changes bind b12 but do so very slowly. This observation is consistent were limited to increased protection at the centre of V3 and a with the lack of neutralizing activity seen with assays that small decrease for V2 residues 176–179 (Fig. 3b). The affected involve a short (30–60 min) incubation period between b12 regions are proximal to the expected PGT123 binding site, as and the BG505 virus assessed from the electron microscopy and X-ray crystal structures of the complex between the BG505 SOSIP.664 trimer and the closely related bNAb, PGT122 (ref. 41) (Fig. 3d). CD4i antibody binding to SOSIP.664 trimers probed by HDX. Antibody 2G12 has evolved a unique domain swap to The HDX profile of the fully formed complex between 17b and enable both Fabs arms to bind to one glycan cluster on gp120 the KNH1144 SOSIP.664 trimers showed that extensive changes (ref. 42). For this reason, 2G12 binding was examined using intact had occurred throughout the Env protein (Fig. 5a,c). Of note is IgG rather than the Fab fragment. The exchange profile showed that most of the changes are similar to those seen on sCD4 that 2G12 binding did not change the protection of the binding (Fig. 6a and Supplementary Fig. 4c), including increased underlying protein structure at any site in the trimer (Fig. 3c,f), protection throughout portions of the gp120 inner domain and even though the complex was fully formed (Supplementary HR1, and the loss in protection for V1/V2, V3 and the FPPR. Fig. 3a). However, unlike with sCD4, all four strands of the bridging sheet became more protected with 17b binding and no significant change was seen in V5. Slow binding of the CD4i antibody, 17b. Unlike the highly The sCD4 complex and the ternary complex with sCD4 potent bNAbs, the CD4i antibody 17b bound very slowly to the and 17b were compared, to assess the changes imparted by 17b to SOSIP.664 trimers. For the KNH1144 trimers, extended incuba- the CD4-bound conformation of the KNH1144 SOSIP.664 tions of up to 72 h were necessary to generate a homogeneous trimer. The addition of 17b had predominantly local effects near complex, while no binding of the 17b Fab to their BG505 its binding site, with the four strands of the bridging sheet, each counterparts could be detected after even this prolonged period becoming substantially more protected (Fig. 5b,d). A slight (Fig. 4a,b). In contrast, soluble two-domain CD4 (sCD4) bound increase in protection was also evident throughout layer 1 of the efficiently to both SOSIP.664 trimers within minutes, as did 17b inner domain and the CD4 binding loop was somewhat more to the CD4-complexed trimers. We note that blue native– protected within the ternary complex than with sCD4 alone. polyacrylamide gel electrophoresis (BN–PAGE) gels show only Finally, there was a small, but reproducible increase in protection two predominant bands rather than a ladder during the forma- for residues 642–645 of gp41 HR2. tion of the 17b complex with unliganded KNH1144 SOSIP.664 trimers, suggesting that when binding does occur it is a concerted event involving all three protomers (Fig. 4b). Discussion The kinetics of complex formation with 17b were also probed The collective comparison of the HDX profiles of SOSIP.664 by HDX using a pulse-labelling strategy. Unlike continuous trimers in complex with different classes of bNAbs reveals the 4 NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ARTICLE N160 BG505 ± PG9 Fab V2 V1 V2 V3 V4 V5 FP HR1 HR2 V1 V3 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h V2 50 100 150 200 250 300 350 400 450 500 550 600 650 +PG9 Residue number BG505 ± PGT123 Fab N301 V2 V1 V2 V3 V4 V5 FP HR1 HR2 V1 N332 V3 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h V3 V2 50 100 150 200 250 300 350 400 450 500 550 600 650 +PGT123 Residue number KNH1144 ± 2G12 IgG V1 V2 V3 V4 V5 FP HR1 HR2 50 100 150 200 250 300 350 400 450 500 550 600 650 V4 Residue number 3 s 1 min 30 min 20 h 50 100 150 200 250 300 350 400 450 500 550 600 650 +2G12 Residue number Figure 3 | Effects of binding of glycan-dependent bNAbs. Butterfly plots show the exchange profiles of SOSIP.664 trimers either unliganded or as complexes with (a) PG9 Fab, (b) PGT123 Fab and (c) 2G12 IgG. The differences are mapped onto the trimer crystal structure (d–f), as described in Fig. 2. As a reference, the approximate positions of the Fabs are shown as transparent surfaces (modelled from PDB accession codes 3NGB, 3U4E and 4NCO) , 2,28,30 while glycans known to be relevant to the epitopes are shown as magenta sticks . No structure is yet available for the PGT123 trimer complex; hence, 2,10 PGT122 is shown instead, as its epitope is very similar (PDB: 4NCO) extent of allosteric effects imparted by each interaction. With the not observe peptides that included residues 129–164 and 326–369 most potent neutralizing antibodies studied here, the changes on we could not monitor changes in those regions (Supplementary bNAb binding are limited to increased protection at the Fig. 2). Despite this limitation, the comparisons definitively show immediate contact regions (Figs 2 and 3). The highly potent that the glycan-dependent antibodies do not exert long-range CD4bs antibodies, VRC01 and PGV04, and all the glycan- effects on the protein framework of SOSIP.664 trimers. dependent antibodies had only limited, focused effects on the One peculiarity of PG9 is that it binds asymmetrically to Env HDX profiles of the trimers. The glycan cluster-targeting 2G12 trimers with a stoichiometry of 1:1 (Fab:trimer) . Such antibody had no effects at all on the underlying protein backbone, asymmetric binding would result in complex exchange kinetics, implying that this epitope does indeed involve only glycans. It as the three lobes of the trimer would not be affected equally. We remains possible that V4, which is proximal to the 2G12 epitope, observed a subtle broadening in the mass spectrum specifically at undergoes some changes, but this loop could not be monitored by V2 within the PG9-bound SOSIP.664 trimer, consistent with the HDX due to its heavy glycosylation. Similarly, PG9 and PGT123 existence of two V2 conformational subpopulations. No such binding may affect more areas of V1/V2 and V3, but as we could effect was seen with unliganded trimers or any other NAb NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 5 & 2015 Macmillan Publishers Limited. All rights reserved. 2G12 Δ% Exchange % Exchange Δ% Exchange % Exchange Δ% Exchange % Exchange +2G12 Free +PGT123 Free +PG9 Free No data More protected No change Less protected ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 (Fig. 2d). This outcome is in direct contrast to the purely local effects that were observed with the much more potent CD4bs bNAbs VRC01 and PGV04. Such an unexpected effect probably results from b12 binding to and stabilizing a trimer conformation that has less ordered secondary structure, resulting in faster amide exchange kinetics despite the decreased solvent accessi- bility arising from solvent occlusion from the protein–protein interface. The binding of b12 also yielded a subtle increase in 1,084 protection for residues 176–179 within V2 (sequence FYRL), something that was not observed with any of the other antibodies (Fig. 6). A structural alignment to the b12 Fab-gp120 core crystal structure indicated that a portion of the b12 paratope on the 480 trimer is adjacent to this region of V2 (Fig. 2d). Hence, residues 176–179, a region absent from the construct used to generate the BG505 b12-gp120 core crystal structure, may contribute directly to the b12 epitope. This notion is supported by the substantial effects that mutations involving residues 176–180 have on both b12 7,44,45 binding and neutralization . It is noteworthy that 17b can bind to KNH1144 SOSIP.664 trimers, but not their BG505 counterparts, in the absence of CD4. The CD4i epitope is not accessible on the unliganded pre-fusion 10,11 BG505 SOSIP.664 trimers . How, then, can 17b bind to the 1,084 corresponding KNH1144 trimers, albeit with very slow kinetics? As 17b binding in the presence of sCD4 or NBD-556 occurs rapidly, the observed slow binding to the closed trimers (that is, no CD4) cannot simply be explained by a slow intrinsic on-rate (k ). We propose that Env trimers, even in the absence of CD4, on undergo transient structural fluctuations to sample an open state, KNH1144 and in doing so the CD4i epitope is formed and exposed. This hypothesis would explain the slow binding kinetics of 17b to the unliganded KNH1144 SOSIP.664 trimers; specifically, 17b binds DMSO BMS-806 NBD-556 only to the transiently open state of the trimers, forming a complex primarily via conformational selection (Fig. 7a). This concept is also consistent with the coordinated binding of three 17b Fabs per trimer (that is, the lack of evidence for partial occupancy). Thus, once opened, the binding of 17b to a single lobe of the trimer blocks the re-formation of the quaternary interactions at the apex, which means that the other two lobes remain accessible for the rapid binding of two more 17b Fabs. In contrast to 17b, CD4 binds rapidly to the trimer, suggesting that major conformational transitions do not need to occur before binding. Instead, it is the CD4 binding event itself that most probably leads to large-scale conformational changes in the WB α Env trimer (Figs 4 and 7a). Figure 4 | Kinetics of 17b Fab binding to SOSIP.664 trimers. (a) BG505 The argument that 17b recognizes an open form of Env trimers and (b) KNH1144 SOSIP.664 trimers were incubated with ligands for is further supported by the HDX comparisons. All the major various times (from 3 min to 72 h) and resolved by BN–PAGE. (c) 17b was changes associated with the opening of the trimer, including loss incubated with KNH1144 SOSIP.664 trimers for various times, the of protection at V1/V2 and V3, were observed in the 17b complex complexes captured by protein G beads, resolved by SDS–PAGE and (Fig. 6). There were only two major differences between the 17b- visualized by immunoblotting. Identical experiments performed in the and sCD4-bound data sets: (1) for 17b, there was increased presence of BMS-806, NBD-556 and sCD4 reveal that NBD-556 and sCD4 protection throughout the bridging sheet, which is in direct both accelerate 17b binding, while BMS-806 blocks it altogether. The gp120 contact with the Fab and (2) for sCD4, there was more protection band derived from SOSIP.664 trimers is in the highlighted region marked by at V5, which is involved in the binding interface for sCD4 but not the arrow. The other bands represent nonspecific binding of the anti-human 17b. When 17b binds to the already opened sCD4-SOSIP.664 serum used as the secondary antibody. trimer complex, the major changes involve stabilization of the regions in direct contact with the Fab (Fig. 5d). We noted that the binding of b12 also caused changes throughout the trimer, with complex. Qualitatively, the degree of protection indicates that a portions of V1/V2 and V3 losing protection. Together with the large proportion of the V2 subdomains become more protected in slow binding of b12 Fab to the BG505 SOSIP.664 trimers, this the PG9 trimer complex. The stabilizing effect of a single bound finding suggests that similar to 17b, b12 recognizes an open PG9 Fab therefore probably extends beyond a single lobe of the conformation of the trimer. trimer, which is consistent with our earlier proposal that the The current study demonstrates that highly potent bNAbs binding interface involves two protomers of the trimer . target the closed conformation of Env without inducing The less potent CD4bs bNAb b12 had a unique effect in that it conformational changes, something that has been observed for resulted in changes throughout the KNH1144 SOSIP.664 trimer, several other Ab–pathogen systems . Less-potent antibodies (for with a loss of protection within some of the contact surface example, b12) may require the trimer to transiently sample 6 NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Unliganded +sCD4 (3 m) +sCD4 & 17b (3 m) +17b (3 m) +17b (4 h) +17b (72 h) +b12 (3 m) +b12 (4 h) +sCD4 (3 m) +b12 (72 h) +17b (3 m) +17b (3 m) +17b (10 m) +17b (4 h) +17b (72 h) (+) Unliganded NC 1 h 12 h 72 h 1 h 12 h 72 h 1 h 12 h 72 h 1 h sCD4 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ARTICLE KNH1144 ± 17b Fab V1 V2 V3 V4 V5 FP HR1 HR2 V2 V1 CD4-binding loop V3 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h Bridging sheet Layer 2 Layer 3 CD4 loop * HR1 Layer 3 Layer 1 FPPR V3 V1/V2 50 100 150 200 250 300 350 400 450 500 550 600 650 +17b HR1 FPPR Residue number KNH1144-sCD4 ± 17b Fab V1 V2 V3 V4 V5 FP HR1 HR2 V3 CD4-binding 0 loop V1/V2 V4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number 3 s 1 min 30 min 20 h Bridging sheet CD4 loop HR2 Bridging Layer 1 sheet +17b and sCD4 50 100 150 200 250 300 350 400 450 500 550 600 650 Residue number Figure 5 | Effects of CD4i antibody binding. The butterfly and difference plots compare unliganded SOSIP.664 trimers (isolate KNH1144) with (a) the binary complex with 17b Fab and (b) the ternary complex with sCD4 and 17b Fab. (c) The differences are mapped onto the BG505 SOSIP.664 trimer crystal structure (PDB accession code: 4NCO), as described in Fig. 2. Contacts with CD4 (yellow) and Fabs (green) are shown as transparent surfaces (modelled relative to the gp120 core from PDB: 1GC1) . The regions unresolved in the trimer structure are shown as dashed lines. (d) The effects of 17b binding to sCD4-bound SOSIP.664 trimers are similarly mapped onto the CD4-bound conformation of gp120, as modelled into the trimer structure (PDB: 3JWD) . The change within HR2 in the ternary complex is not shown, as this region is not resolved in the 4NCO trimer structure. 100 100 100 100 50 50 50 50 (V1) (V2) (V3) (V2) (149–159) (164–175) (288–317) (166–175) KEMEGEIKNCSF EIRDKKQKVYSL LVEPVRI...VRIGPGQAF RDKKQKVYSL 0 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 110 10 10 10 10 110 10 10 10 10 110 10 10 10 10 110 10 10 10 10 Time (s) Time (s) Time (s) Time (s) 100 100 100 100 V2 50 50 50 50 (V2) (180–193) (V3) (V3) (176–179) DVVPINQGNSS... (317–324) (317–325) FYRL ...SKNSSEYRL FATGDIIG YATGDIIGD 0 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 110 10 10 10 10 110 10 10 10 10 110 10 10 10 10 110 10 10 10 10 Time (s) Time (s) Time (s) Time (s) Trimer only sCD4 b12 VRC01 PGT123 2G12 17b sCD4 and 17b PGV04 PG9 Figure 6 | Individual exchange profiles of peptides covering V1/V2 and V3. The plots for selected peptides derived from (a) KNH1144 and (b) BG505 SOSIP.664 trimers in unliganded trimer or in complex with sCD4 or various antibodies, as stated. Dots show the mean uptake values at each time point with error bars showing the s.d. between duplicate measurements (in many cases, the error bars are too small to see). Plots for all observable peptides are shown in Supplementary Figs 1 and 2. NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 7 & 2015 Macmillan Publishers Limited. All rights reserved. % Exchanged % Exchanged Δ% Exchange Δ% Exchange % Exchange % Exchange % Exchanged % Exchanged % Exchanged % Exchanged +17b +17b Free +sCD4 and sCD4 % Exchanged % Exchanged No data More protected No change Less protected A ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 10–12,19,47 bridging sheet . Moreover, quaternary steric hindrance effects restrict how b12 can gain access to the CD4 binding site on 9,17 the closed, pre-fusion trimer , a constraint that is absent from Env Env closed open monomeric gp120. The highly ordered, unliganded conformation of the Env 17b CD4 trimer in which the trimer apex is formed by the V1/V2 and V3 loops, and in which conserved epitopes such as the co-receptor Env ·CD4 Env ·17b closed open binding site are masked, is often referred to as the ‘closed’ conformation or ‘ground state’ . The binding of CD4 induces profound allosteric changes such that the trimer adopts an ‘open’ Env ·CD4 configuration, with the quaternary associations between V1/V2 open and V3 at the trimer apex now released. Cryo-electron tomography images of the unliganded (closed) and CD4-bound (fully open) trimer provide a low-resolution, but highly Tier 2+ Tier 1 CD4 independent 9,17,18 informative, view of these two states . We note that a much earlier usage of the ‘open/closed’ terminology refers to the conformations of Env on viruses, as inferred from their neutralization phenotypes. In the 1990s, it was common to refer to neutralization-sensitive (usually cell line adapted) viruses as ‘Closed’ ‘Open’ having a more open (that is, antibody accessible) form of Env gp120 gp41 V1/V2 V3 49–52 than their resistant, primary isolate counterparts . The phenotypic difference between the neutralization-sensitive, cell Figure 7 | Conformational sampling of unliganded Env trimers. (a) CD4 line-adapted, tier 1 isolates and more resistant primary isolates can bind the closed form of Env and allosterically induce an open (now categorized as tier 2 or 3) has been attributed to undefined conformation. In contrast, 17b can only bind to unliganded Env when the 7,26,53,54 aspects of the quaternary structure of their trimers . open conformation is transiently sampled, and by doing so it stabilizes this A very recent paper emphasizes the critical role of the V2 loop in conformation. (b) Env trimers exist predominantly in the closed form in these neutralization phenotypes, probably via influences on the which the interactions between V1/V2 from each protomer are intact and formation and stability of the inter-protomer associations at the V3 is occluded. Transient sampling of open conformations disrupts the trimer apex . inter-protomer interactions at the trimer apex. The trimers on higher tier Here we propose that the subtly differing open/closed (more neutralization-resistant) viruses are less likely to sample open terminologies may be manifestations of a single phenomenon: conformations, thereby protecting conserved epitopes from NAbs. Tier 1 the transient opening motions of the trimer that occur at different primary and cell line-adapted isolates sample the open conformation more rates among different isolates that are a major determinant often or for longer periods, which renders these viruses more vulnerable to of neutralization susceptibility (Fig. 7b). Under the immune neutralization. CD4-independent isolates (which are extremely pressures that target conserved regions such as the co-receptor neutralization sensitive) have evolved mutations that stabilize the open binding site, the more neutralization-resistant isolates have state, probably to facilitate co-receptor binding when CD4 is either present evolved Env trimers that better mask conserved epitopes by at low levels or entirely absent. sampling the open conformations only transiently and infre- 48–50,56,57 quently . Conversely, cell line-adapted isolates that were cultured in the absence of antibody selection pressures evolved to partially open states in which their epitopes are exposed. sample the open state more readily. CD4-independent isolates, Antibodies such as 17b whose epitopes are masked in the which can enter cells that lack CD4 but express the co-receptor, 58,59 closed trimer conformation are likely to recognize only the fully are extremely susceptible to neutralization and primarily exist open trimer conformation in which the co-receptor binding site is in the open state . This trend is consistent with the recent unmasked and its elements assembled. This trend is consistent observation that a higher level of ‘intrinsic reactivity’ (ability of with low resolution cryo-electron tomography studies, which Env to sample different conformations) correlates with sensitivity reported that sCD4 and 17b binding resulted in open trimer to antibody neutralization . Our study is limited to the conformations, that b12 binding caused the trimer to partially KNH1144 and BG505 SOSIP.664 constructs, which represent 9,17,18 open, and that VRC01 binding caused no such opening . the only soluble native-like Env trimers currently available. Furthermore, both CD4 and b12 have been observed to induce Examination of native-like trimers from other isolates will be gp120 shedding from virus, a presumed marker of substantial necessary for a complete understanding of the relationship conformational changes, whereas VRC01 and PGV04 do not have between the kinetics of Env’s transient opening motions and 45,46 such an effect . neutralization susceptibility. It is relevant to draw a distinction between what happens when Although our HDX analysis has revealed much about the NAbs bind to the gp120 subunits of trimers compared with conformational state of the trimer, under the current conditions it individual gp120 monomers. For example, VRC01 binding to does not appear to provide a quantitative measure of the kinetics gp120 monomers is associated with a large entropic penalty, of transient opening motions. If the open conformations persisted suggesting that gp120 transitions from a highly dynamic in solution on a long timescale (seconds), then it would result in form to a more constrained conformation . In contrast, b12 correlated exchange kinetics (EX1 regime) as evidenced by binding to monomeric gp120 is associated with a much less bimodal mass spectra under our experimental conditions . significant change in entropy, suggestive of a less profound However, no such EX1 behaviour was observed for the structural change . This pattern is the inverse of what we see unliganded trimer and all portions of the protein exchange when the same two NAbs interact with the trimer; the probable through fast local structural fluctuations (EX2). Furthermore, explanation is that gp120 in the trimer context is more ligands that are expected to stabilize the closed conformation (for conformationally constrained then when it is an isolated example, PG9) only show local effects at their binding site, monomer, particularly with respect to V1/V2, V3 and the without altering the HDX profile through the rest of the trimer 8 NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ARTICLE apex. Likewise, under the experimental conditions used, Hydrogen/deuterium exchange. SOSIP.664–antibody complexes (15 mg) were diluted tenfold into deuterated PBS buffer and incubated at room temperature. SOSIP.664 from BG505 and KNH1144 isolates had very similar Aliquots removed after 3 s, 1 min, 30 min and 20 h were quenched by mixing with HDX profiles, despite phenotypic differences. For one, KNH1144 an equal volume of cold 200 mM Tris-2-carboxyethyl phosphine, 0.2% formic acid 19,32 has lower thermal stability than BG505 trimers . The kinetics (final pH 2.5). The samples were subsequently digested with pepsin (at of b12 and 17b binding are also drastically different between the 0.15 mg ml ) for 5 min on ice, flash frozen in liquid nitrogen and stored at 80 C. For liquid chromatography–MS analysis, samples were thawed on ice for two isolates, indicating a lower frequency of exposure of several 5 min and manually injected onto a Waters BEH 1.7 mm, 1.2 5 mm trap column epitopes in BG505. The relative kinetics of ligand binding to the (Waters) flowing 0.1% TFA at 200mlmin . After 3 min of washing, the peptides partially open (with b12) or fully open (with 17b) form of the were resolved over a Hypersil 1 50 mm, 2.1 mm C18 column (Thermo Scientific), trimer should serve as an additional assay for assessing the degree using a gradient of 15%–40% B in 8 min (A: 0.05% TFA, 5% ACN; B: 0.05% TFA, 80% ACN). Two hundred microlitres of 10% formic acid, 80% MeOH, 2:1 of transient opening motions within different Env trimers. IPA:ACN and 80% ACN were injected over the loop and trap column between Recently, single-molecule fluorescence experiments have injections, while two rapid (10%–100% B in 30 s) gradients were used to clean the demonstrated that native Env on the membrane samples transient resolving column, to minimize carryover. Eluting peptides were analysed with a open states, and that potent neutralizing antibodies stabilize the Waters Synapt Q-TOF mass spectrometer, with source and desolvation tempera- 12,48 closed state of the trimer . Furthermore, in agreement with tures of 80C and 150 C, respectively. Peptic peptides were identified by exact mass and MS/MS spectra. Identification our own observations, the transient fluctuations to the activated of glycopeptides was aided by MS/MS spectra of the fully deglycosylated samples as (open) states were more frequent with neutralization-sensitive described previously . Per cent exchange for each time point was calculated isolates. Despite any caveats that may be associated with the use relative to zero and fully deuterated standards as described previously . For ligand of soluble SOSIP.664 trimers (that is, the presence of stabilizing binding kinetics, SOSIP.664 trimers at 1 mg ml were incubated with a threefold excess of b12 or 17b Fab in PBS at 22 C. At 3 min, 4 h and 72 h, an aliquot of the mutations and the absence of the MPER/transmembrane mixture was diluted tenfold into deuterated PBS buffer for 3 s and rapidly segments of gp41), their properties and behaviour do appear to quenched. reflect what is observed using full-length, fusion-competent Env trimers. Beyond the concurrence in behaviours between soluble SOSIP.664 trimers and Env on virus, characterizing the Antibody binding assays. The kinetics of 17b binding were measured by incu- interaction of the SOSIP trimer with antibodies provides bating SOSIP.664 trimers at 1 mg ml with a threefold molar excess of 17b Fab (relative to protomers) and a monitoring complex formation by BN–PAGE. valuable information for understanding its properties as a Immunoprecipitation experiments were initiated by addition of 2 mg of 17b Fab to potential immunogen. 3 mg of KNH1144 SOSIP.664 trimers in 120ml of PBS plus 2% dimethyl sulfoxide, The development of broadly cross-reactive antibodies is together with the following: no ligand, 100mM NBD-556, 100mM BMS-806 or relatively uncommon during the course of HIV-1 infection and 0.4 mM of sCD4. Pre-washed protein G sepharose beads were added after the mixtures were incubated at 25 C for various times, and rotated on a nutator for generally takes several years when it does happen . In light 1 h. The beads were pelleted by centrifugation and washed four times in PBS plus of the fact that all of the highly potent bNAbs target the closed 0.1% Tween 20, followed by re-suspension and boiling in reducing SDS-gel loading form of Env, more potent Ab responses may be achieved by buffer (Invitrogen). Western blottings were probed with HIV-Ig serum (1:1,000 using trimer immunogens that present the closed form of dilution), followed by anti-human-horseradish peroxidase (1:5,000 dilution; Jack- son Immunoresearch). The labelled bands were visualized by chemilluminescence Env as stably as possible. For such immunogens, any transient (Western lightning, Perkin Elmer). sampling of open conformations should be minimized to avoid exposing undesirable, non-neutralizing epitopes that could potentially distract the humoral immune response. The BG505 References SOSIP.664 protein appears to embody such an immunogen 1. Walker, L. M. & Burton, D. R. Rational antibody-based HIV-1 vaccine design: that presents the major neutralizing epitopes in the context current approaches and future directions. Curr. Opin. Immunol. 22, 358–366 of a stable, closed trimer . The contrast between the KNH1144 (2010). 2. Walker, L. M. et al. Broad neutralization coverage of HIV by multiple highly and BG505 SOSIP.664 trimers, which bind 17b slowly versus not potent antibodies. Nature 477, 466–470 (2011). at all, implies that the BG505 trimers are the more likely to 3. Mascola, J. R. & Haynes, B. F. HIV-1 neutralizing antibodies: understanding remain in the fully closed state, sampling the open state(s) less nature’s pathways. Immunol. Rev. 254, 225–244 (2013). frequently than their KNH1144 counterparts. Further 4. Wyatt, R. et al. The antigenic structure of the HIV gp120 envelope modifications to the BG505 SOSIP.664 trimers may be useful glycoprotein. Nature 393, 705–711 (1998). 5. Kwong, P. D. et al. HIV-1 evades antibody-mediated neutralization through for reducing the spontaneous or CD4-induced opening of the conformational masking of receptor-binding sites. Nature 420, 678–682 (2002). trimer that is likely to occur in vivo during the course of 6. Wei, X. et al. Antibody neutralization and escape by HIV-1. Nature 422, vaccination. Identifying other closed conformation, native-like 307–312 (2003). trimers with comparable properties is an additional priority, not 7. Pantophlet, R. et al. Fine mapping of the interaction of neutralizing and least to allow the exploration of more complex immunization nonneutralizing monoclonal antibodies with the CD4 binding site of human regimens. immunodeficiency virus type 1 gp120. J. Virol. 77, 642–658 (2003). 8. Wu, X. et al. Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing. Science 333, 1593–1602 (2011). 9. Liu, J., Bartesaghi, A., Borgnia, M. J., Sapiro, G. & Subramaniam, S. Molecular Methods architecture of native HIV-1 gp120 trimers. Nature 455, 109–113 (2008). Sample preparation. SOSIP.664 trimers based on the subtype A isolates 10. Julien, J. P. et al. Crystal structure of a soluble cleaved HIV-1 envelope trimer. KNH1144 and BG505 were expressed in 293S GnTI / cells and purified by Science 342, 1477–1483 (2013). 2G12 affinity followed by size-exclusion chromatography (Superdex S200, GE 11. Lyumkis, D. et al. Cryo-EM structure of a fully glycosylated soluble cleaved Healthcare) in PBS (20 mM sodium phosphate pH 7.4, 150 mM NaCl with 1 mM HIV-1 envelope trimer. Science 342, 1484–1490 (2013). 19,63 EDTA and 0.02% sodium azide) as described previously . sCD4 (ref. 64), HIV- 12. Pancera, M. et al. Structure and immune recognition of trimeric pre-fusion 4,14,23,24 IG and monoclonal antibodies 17b , b12 (refs 20,65,66), 2G12 (refs 27,67) HIV-1 Env. Nature 514, 455–461 (2014). and VRC01 (ref. 68) were obtained from the NIH AIDS reagents programme. Fabs 13. Wu, L. et al. CD4-induced interaction of primary HIV-1 gp120 glycoproteins were prepared using Pierce Fab preparation kit (Thermo Scientific) according to with the chemokine receptor CCR-5. Nature 384, 179–183 (1996). manufacturer’s instructions. PGV04, PGT123 and PG9 Fabs were prepared as 69 14. Trkola, A. et al. CD4-dependent, antibody-sensitive interactions between HIV- described previously . All proteins were buffer exchanged into PBS. Complexes 1 and its co-receptor CCR-5. Nature 384, 184–187 (1996). with SOSIP.664 trimers were formed by overnight incubation with a threefold 15. Rizzuto, C. D. et al. A conserved HIV gp120 glycoprotein structure involved in molar excess of ligand (relative to each protomer) at 4 C, except for the 17b Fab chemokine receptor binding. Science 280, 1949–1953 (1998). complex, which was incubated for 72 h at room temperature. SDS– and BN–PAGE analyses were performed with the same samples used for HDX, to ensure that 16. White, T. A. et al. Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary complexes had formed and that no degradation had occurred throughout the incubations (Supplementary Fig. 3). structure. PLoS Pathog. 6, e1001249 (2010). NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 9 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 17. Tran, E. E. et al. Structural mechanism of trimeric HIV-1 envelope glycoprotein 47. Guttman, M., Kahn, M., Garcia, N. K., Hu, S. L. & Lee, K. K. Solution structure, activation. PLoS Pathog. 8, e1002797 (2012). conformational dynamics, and CD4-induced activation in full-length, 18. Harris, A. et al. Trimeric HIV-1 glycoprotein gp140 immunogens and native glycosylated, monomeric HIV gp120. J. Virol. 86, 8750–8764 (2012). HIV-1 envelope glycoproteins display the same closed and open quaternary 48. Munro, J. B. et al. Conformational dynamics of single HIV-1 envelope trimers molecular architectures. Proc. Natl Acad. Sci. USA 108, 11440–11445 (2011). on the surface of native virions. Science 346, 759–763 (2014). 19. Guttman, M. et al. CD4-induced activation in a soluble HIV-1 Env trimer. 49. Moore, J. P., Trkola, A., Sattentau, Q. J., Cao, Y. & Ho, D. D. Studies on the Structure 22, 974–984 (2014). interactions of monoclonal antibodies with HIV-1 isolates from clades A-F. 20. Burton, D. R. et al. Efficient neutralization of primary isolates of HIV-1 by a Proceedings of IX Colloque des Cent Gardes 151–155 (1994). recombinant human monoclonal antibody. Science 266, 1024–1027 (1994). 50. Moore, J. P., Sattentau, Q. J., Wyatt, R. & Sodroski, J. Probing the structure of 21. Zhou, T. et al. Structural definition of a conserved neutralization epitope on the human immunodeficiency virus surface glycoprotein gp120 with a panel of HIV-1 gp120. Nature 445, 732–737 (2007). monoclonal antibodies. J. Virol. 68, 469–484 (1994). 22. Zhou, T. et al. Structural basis for broad and potent neutralization of HIV-1 by 51. Sullivan, N., Sun, Y., Li, J., Hofmann, W. & Sodroski, J. Replicative function and antibody VRC01. Science 329, 811–817 (2010). neutralization sensitivity of envelope glycoproteins from primary and T-cell 23. Thali, M. et al. Characterization of conserved human immunodeficiency line-passaged human immunodeficiency virus type 1 isolates. J. Virol. 69, virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. 4413–4422 (1995). J. Virol. 67, 3978–3988 (1993). 52. Sattentau, Q. J. & Moore, J. P. Human immunodeficiency virus type 1 24. Kwong, P. D. et al. Structure of an HIV gp120 envelope glycoprotein in neutralization is determined by epitope exposure on the gp120 oligomer. J. Exp. complex with the CD4 receptor and a neutralizing human antibody. Nature Med. 182, 185–196 (1995). 393, 648–659 (1998). 53. Fouts, T. R., Binley, J. M., Trkola, A., Robinson, J. E. & Moore, J. P. 25. Salzwedel, K., Smith, E. D., Dey, B. & Berger, E. A. Sequential CD4-coreceptor Neutralization of the human immunodeficiency virus type 1 primary isolate JR- interactions in human immunodeficiency virus type 1 Env function: soluble FL by human monoclonal antibodies correlates with antibody binding to the CD4 activates Env for coreceptor-dependent fusion and reveals blocking oligomeric form of the envelope glycoprotein complex. J. Virol. 71, 2779–2785 activities of antibodies against cryptic conserved epitopes on gp120. J. Virol. 74, (1997). 326–333 (2000). 54. Kwong, P. D. et al. Structures of HIV-1 gp120 envelope glycoproteins from 26. Seaman, M. S. et al. Tiered categorization of a diverse panel of HIV-1 Env laboratory-adapted and primary isolates. Structure 8, 1329–1339 (2000). pseudoviruses for assessment of neutralizing antibodies. J. Virol. 84, 1439–1452 55. Brandenberg, O. F. et al. Partial rescue of V1V2 mutant infectivity by HIV-1 (2010). cell-cell transmission supports the domain’s exceptional capacity for sequence 27. Trkola, A. et al. Human monoclonal antibody 2G12 defines a distinctive variation. Retrovirology 11, 75 (2014). neutralization epitope on the gp120 glycoprotein of human immunodeficiency 56. Kolchinsky, P., Kiprilov, E. & Sodroski, J. Increased neutralization sensitivity of virus type 1. J. Virol. 70, 1100–1108 (1996). CD4-independent human immunodeficiency virus variants. J. Virol. 75, 28. Sanders, R. W. et al. The mannose-dependent epitope for neutralizing antibody 2041–2050 (2001). 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J. Virol. 57. Kabat, D., Kozak, S. L., Wehrly, K. & Chesebro, B. Differences in CD4 76, 7293–7305 (2002). dependence for infectivity of laboratory-adapted and primary patient 29. Murin, C. D. et al. Structure of 2G12 Fab2 in complex with soluble and fully isolates of human immunodeficiency virus type 1. J. Virol. 68, 2570–2577 glycosylated HIV-1 Env by negative-stain single particle electron microscopy. (1994). J. Virol. 88, 10177–10188 (2014). 58. Hoffman, T. L. et al. Stable exposure of the coreceptor-binding site in a CD4- 30. Walker, L. M. et al. Broad and potent neutralizing antibodies from an African independent HIV-1 envelope protein. Proc. Natl Acad. Sci. USA 96, 6359–6364 donor reveal a new HIV-1 vaccine target. Science 326, 285–289 (2009). (1999). 31. McLellan, J. S. et al. Structure of HIV-1 gp120 V1/V2 domain with broadly 59. Edwards, T. G. et al. Relationships between CD4 independence, neutralization neutralizing antibody PG9. Nature 480, 336–343 (2011). sensitivity, and exposure of a CD4-induced epitope in a human 32. Julien, J. P. et al. Asymmetric recognition of the HIV-1 trimer by broadly immunodeficiency virus type 1 envelope protein. J. Virol. 75, 5230–5239 neutralizing antibody PG9. Proc. Natl Acad. Sci. USA 110, 4351–4356 (2013). (2001). 33. Bartesaghi, A., Merk, A., Borgnia, M. J., Milne, J. L. & Subramaniam, S. 60. Haim, H. et al. Contribution of intrinsic reactivity of the HIV-1 envelope Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by glycoproteins to CD4-independent infection and global inhibitor sensitivity. cryo-electron microscopy. Nat. Struct. Mol. Biol. 20, 1352–1357 (2013). PLoS Pathog. 7, e1002101 (2011). 34. Blattner, C. et al. Structural delineation of a quaternary, cleavage-dependent 61. Weis, D. D., Wales, T. E., Engen, J. R., Hotchko, M. & Ten Eyck, L. F. epitope at the gp41-gp120 interface on intact HIV-1 Env trimers. Immunity 40, Identification and characterization of EX1 kinetics in H/D exchange mass 669–680 (2014). spectrometry by peak width analysis. J. Am. Soc. Mass Spectrom. 17, 1498–1509 35. Klasse, P. J. & Sattentau, Q. J. Occupancy and mechanism in antibody-mediated (2006). neutralization of animal viruses. J. Gen. Virol. 83, 2091–2108 (2002). 62. Stamatatos, L., Morris, L., Burton, D. R. & Mascola, J. R. Neutralizing 36. Reading, S. A. & Dimmock, N. J. Neutralization of animal virus infectivity by antibodies generated during natural HIV-1 infection: good news for an HIV-1 antibody. Arch. Virol. 152, 1047–1059 (2007). vaccine? Nat. Med. 15, 866–870 (2009). 37. Sanders, R. W. et al. A next-generation cleaved, soluble HIV-1 Env 63. Iyer, S. P. et al. Purified, proteolytically mature HIV type 1 SOSIP gp140 trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly envelope trimers. AIDS Res. Hum. Retroviruses 23, 817–828 (2007). neutralizing but not non-neutralizing antibodies. PLoS Pathog. 9, e1003618 64. Garlick, R. L., Kirschner, R. J., Eckenrode, F. M., Tarpley, W. G. & (2013). Tomich, C. S. Escherichia coli expression, purification, and biological 38. Marcsisin, S. R. & Engen, J. R. Hydrogen exchange mass spectrometry: what is activity of a truncated soluble CD4. AIDS Res. Hum. Retroviruses 6, 465–479 it and what can it tell us? Anal. Bioanal. Chem. 397, 967–972 (2010). (1990). 39. Eroshkin, A. M. et al. bNAber: database of broadly neutralizing HIV antibodies. 65. Burton, D. R. et al. A large array of human monoclonal antibodies to type 1 Nucleic Acids Res. 42, D1133–D1139 (2014). human immunodeficiency virus from combinatorial libraries of asymptomatic 40. Ringe, R. P. et al. Cleavage strongly influences whether soluble HIV-1 envelope seropositive individuals. Proc. Natl Acad. Sci. USA 88, 10134–10137 glycoprotein trimers adopt a native-like conformation. Proc. Natl Acad. Sci. (1991). USA 110, 18256–18261 (2013). 66. Barbas, 3rd C. F. et al. Recombinant human Fab fragments neutralize 41. Julien, J. P. et al. Broadly neutralizing antibody PGT121 allosterically modulates human type 1 immunodeficiency virus in vitro. Proc. Natl Acad. Sci. USA 89, CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple 9339–9343 (1992). surrounding glycans. PLoS Pathog. 9, e1003342 (2013). 67. Buchacher, A. et al. Generation of human monoclonal antibodies against 42. Calarese, D. A. et al. Antibody domain exchange is an immunological solution HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for to carbohydrate cluster recognition. Science 300, 2065–2071 (2003). peripheral blood lymphocyte immortalization. AIDS Res. Hum. Retroviruses 10, 43. Schon, A. et al. Thermodynamics of binding of a low-molecular-weight CD4 359–369 (1994). mimetic to HIV-1 gp120. Biochemistry 45, 10973–10980 (2006). 68. Wu, X. et al. Rational design of envelope identifies broadly neutralizing human 44. Mo, H. et al. Human immunodeficiency virus type 1 mutants that escape monoclonal antibodies to HIV-1. Science 329, 856–861 (2010). neutralization by human monoclonal antibody IgG1b12. J. Virol. 71, 69. Pejchal, R. et al. Structure and function of broadly reactive antibody PG16 6869–6874 (1997). reveal an H3 subdomain that mediates potent neutralization of HIV-1. Proc. 45. Li, Y. et al. Mechanism of neutralization by the broadly neutralizing HIV-1 Natl Acad. Sci. USA 107, 11483–11488 (2010). monoclonal antibody VRC01. J. Virol. 85, 8954–8967 (2011). 70. Pancera, M. et al. Structure of HIV-1 gp120 with gp41-interactive region 46. Falkowska, E. et al. PGV04, an HIV-1 gp120 CD4 binding site antibody, is reveals layered envelope architecture and basis of conformational mobility. broad and potent in neutralization but does not induce conformational changes Proc. Natl Acad. Sci. USA 107, 1166–1171 (2010). characteristic of CD4. J. Virol. 86, 4394–4403 (2012). 10 NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7144 ARTICLE Acknowledgements Author contributions The following reagents were obtained through the AIDS Research and Reference Reagent M.G. conducted the experiments. A.C., J.-P.J. and R.W.S. provided essential reagents. Program, Division of AIDS, NIAID, NIH: sCD4-183 from Pharmacia, Inc.; HIV-1 gp120 M.G. and K.K.L. analysed the data and made the figures. M.G., J.-P.J., R.W.S., I.A.W., Monoclonal Antibody (IgG1 b12) from Dr Dennis Burton and Carlos Barbas; HIV-1 gp120 J.P.M. and K.K.L. wrote the manuscript. Monoclonal Antibody (17b) from Dr James E. Robinson; HIV-1 gp120 MAb (VRC01) from Dr John Mascola; HIV-1 gp120 Monoclonal Antibody (2G12) from Additional information Dr Hermann Katinger; HIV-IG from NABI and NHLBI. This work was supported by NIH grant F32-GM097805 (M.G.), R01-GM099989 and R21-AI112389 (K.K.L.), P01- Supplementary Information accompanies this paper at http://www.nature.com/ AI82362 (J.P.M. and I.A.W.), R01-AI084817 (I.A.W.), R37-AI36082 (J.P.M.), UAB CFAR naturecommunications grant (P30-AI027767) through the CNIHR programme for new HIV investigators (K.K.L.), Competing financial interests: The authors declare no competing financial interests. as well as the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Dis- covery (CAVD) grant OPP1033102 (K.K.L.) and the International AIDS Vaccine Initiative Reprints and permission information is available online at http://npg.nature.com/ Neutralizing Antibody Consortium and Center (I.A.W.), CHAVI-ID UM1-AI100663 reprintsandpermissions/ (I.A.W.), a Vidi grant from the Netherlands Organization for Scientific Research (NWO) (R.W.S.), a Starting Investigator Grant from the European Research Council ERC-StG- How to cite this article: Guttman, M. et al. Antibody potency relates to the 2011-280829-SHEV (R.W.S.) and a Canadian Institutes of Health Research fellowship (J.- ability to recognize the closed, pre-fusion form of HIV Env. Nat. Commun. 6:6144 P.J.). We thank Andrew Ward and Per Johan Klasse for helpful comments and suggestions. doi: 10.1038/ncomms7144 (2015). NATURE COMMUNICATIONS | 6:6144 | DOI: 10.1038/ncomms7144 | www.nature.com/naturecommunications 11 & 2015 Macmillan Publishers Limited. All rights reserved.

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Nature Communications – Springer Journals

Published: Feb 5, 2015

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