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. Author manuscript; available in PMC: 2015 May 15.
Published in final edited form as: Immunity. 2014 Apr 24;40(5):669–680. doi: 10.1016/j.immuni.2014.04.008

Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers

Claudia Blattner 1,2,11, Jeong Hyun Lee 1,3,11, Kwinten Sliepen 8, Ronald Derking 8, Emilia Falkowska 2,4, Alba Torrents de la Peña 8, Albert Cupo 9, Jean-Philippe Julien 1,2,3, Marit van Gils 8, Peter S Lee 1, Wenjie Peng 5,6, James C Paulson 5,6, Pascal Poignard 2,4, Dennis R Burton 2,3,4,10, John P Moore 9, Rogier W Sanders 8,9, Ian A Wilson 1,2,3,7,✉, Andrew B Ward 1,2,3,✉
PMCID: PMC4057017  NIHMSID: NIHMS585680  PMID: 24768348

Summary

All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. As PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer.

Introduction

Over the past five years, many new broadly neutralizing antibodies (bnAbs) have been isolated from HIV-1 infected humans that are able to neutralize diverse panels of HIV-1 in vitro (Burton et al., 2012b; Huang et al., 2012; Klein et al., 2013; Kwong and Mascola, 2012; Scheid et al., 2011; Walker et al., 2011; Walker et al., 2009; Zhou et al., 2010). These advances have reinvigorated the pursuit of both active and passive HIV-1 vaccine strategies (Barouch et al., 2013; Burton et al., 2012a; Horwitz et al., 2013; Klein et al., 2012b; Shingai et al., 2013). The sole target for bnAbs are native, functional envelope glycoprotein (Env) trimers on the virus surface. These trimers of gp120 and gp41 heterodimers assemble after endoproteolytic cleavage of the gp160 precursor. Immune selection pressures, combined with a high replication rate and an error-prone reverse transcriptase, create hypervariable HIV-1 Env proteins. The trimer surface is also shielded by an extensive array of glycans. Nonetheless, sites of vulnerability on the virus do exist and four bnAb epitope clusters have been characterized: a linear region of gp41 close to the viral membrane (the membrane proximal external region or MPER) (Huang et al., 2012; Muster et al., 1993; Zwick et al., 2001); the CD4 binding site on gp120 (Burton et al., 1994; Scheid et al., 2011; Wu et al., 2011; Zhang et al., 2012; Zhou et al., 2010); an N332-dependent epitope cluster on the glycosylated face of gp120 (Kong et al., 2013; Walker et al., 2011); and a site including the N160 glycan on V2 at the trimer apex (Julien et al., 2013b; McLellan et al., 2011; Walker et al., 2011; Walker et al., 2009). Another suspected bnAb site on gp120 has also been partially characterized (Klein et al., 2012a; Thali et al., 1993; Xiang et al., 2002; Zhang et al., 2004). All known bnAbs characterized to date bind to one of these sites, raising the question of whether all broadly neutralizing epitopes on Env have already been identified.

Analyzing human responses to viral infection by direct functional screening (Simek et al., 2009; Walker et al., 2011; Walker et al., 2009) has led to the isolation of several potent bnAbs, including a set now designated as the PGT151 family (Falkowska et al., accompanying manuscript). Here, we identify and structurally define the complex quaternary epitope targeted by PGT151 family members and show that it is present only on native-like, cleaved forms of Env. The stability provided to native Env by PGT151 binding creates an opportunity to isolate and purify these trimers from the cell membrane for structural and functional studies. Along with high resolution X-ray crystal structures of the fragment, antigen binding (Fab) of PGT151 and PGT152, we present single particle electron microscopy (EM) reconstructions at ∼19-25 Å resolution of complexes of PGT151 Fab with cell membrane-extracted Env from three different HIV-1 isolates (clade A BG505, clade B JR-FL, and clade C IAVI C22) and compare them with soluble, cleaved SOSIP.664 trimers (Julien et al., 2013b; Julien et al., 2013c; Kong et al., 2013; Sanders et al., 2013). Analyses of the four PGT151-Env structures, in conjunction with higher resolution models of Env (Julien et al., 2013a; Lyumkis et al., 2013) and mutagenesis data, reveal that PGT151 binds an epitope that involves both inter- and intra-protomer contacts with the trimer and is dependent on a subset of fully processed glycans. We also show that PGT151 binds with a unique stoichiometry of 2 Fabs per trimer that is likely linked to its quaternary preference. Thus, PGT151 binds an extensive epitope spanning multiple protein subunits and enables, for the first time, high fidelity assessment and isolation of properly formed HIV-1 Env trimers from membranes.

Results

PGT151 family bnAbs bind only cleaved, trimeric Env and require a complex glycan

In ELISA studies, PGT151 bound strongly to soluble, cleaved BG505 SOSIP.664 trimers expressed in HEK293T cells (Figure 1A). These engineered trimers are stabilized by introduction of a disulfide bond between position 501 in gp120 and position 605 in gp41 (termed SOS), along with an isoleucine to proline substitution at position 559 (termed IP). The SOSIP trimers have a native-like conformation and glycosylation profile that includes both high mannose and more complex structures (Julien et al., 2013b; Sanders et al., 2013; Sanders et al., 2002). PGT151 does not bind monomeric BG505 gp120 and reacts inefficiently with HEK293S cell-expressed trimers that bear only high mannose glycans (Figure 1A). The latter observation is consistent with the preference of PGT151 and PGT152 for tri- and tetra-antennary N-linked glycans in a microarray study (Falkowska et al., 2014). Here, we show that PGT151 and PGT152 react only weakly (KD ≈ 1 mM) with tri-antennary N-linked glycans in an isothermal titration calorimetry (ITC) solution-phase assay (Figure 1B).

Figure 1. PGT151 and PGT152 binding to SOSIP gp140 Env trimer is dependent on glycosylation and cleavage and requires the pre-fusion conformation of gp41.

Figure 1

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(A) PGT151 binds strongly to BG505 SOSIP.664 gp140 trimers produced in untreated HEK 293T cells but not to the same trimers produced in HEK 293S cells (Man5-9 glycans only) or in HEK 293T cells treated with kifunensine (kif) (Man9 glycans only). PGT151 also does not bind BG505 gp120 monomers. 2G12, which binds high mannose glycans, served as a control MAb. 2G12 binding to trimers produced in kifunensine-treated 293T cells or in 293S cells, was modestly increased as a result of the elevated amount of high mannose glycans. The data were derived using a Ni-NTA ELISA and His-tagged Env proteins. Data are representative of at least 2 independent experiments. (B) In an ITC assay, PGT151 and PGT152 Fabs bind with low affinity (KD ≈ 1 mM) to complex type asialo-tri-antennary glycans. The raw binding (top) and integrated titration (bottom) curves are shown. (C) PGT151 binds His-tagged BG505 SOSIP.664 and BG505 IP.664, but not to BG505 WT.664 as shown by Ni-NTA-capture ELISA. Binding of the anti-gp120 MAbs VRC01 and 2G12 to the BG505 IP.664 and BG505 WT.664 variants is greatly reduced (left). The anti-gp41 MAbs F240 and 7B2 do not bind to SOSIP.664 trimers because their epitopes on gp41 are occluded by the gp120 subunits, but they do bind to IP.664 and WT.664 proteins from which gp120 has dissociated (middle). PGT151 binds the SOSIP.664 trimers and the IP.664 proteins, but does not bind cleaved WT.664 (right). These data suggest that the PGT151 epitope is only present in the pre-fusion conformation of gp41. Data presented here were chosen from 2-6 independent experiments. Each experiment was done in duplicate and averaged values are shown.

The PGT151 family of bnAbs is highly specific for cleaved, and thus native-like trimers, whether soluble BG505 SOSIP.664 or embedded in cell membrane (JR-FL) (Falkowska et al., 2014), implying that cleavage causes rearrangements in Env quaternary structure. To explore this property further, we first tested PGT151 binding by ELISA to the His6-tagged BG505 IP.664 construct, which lacks the stabilizing disulfide bond between gp120 and gp41 and, hence, is prone to gp120 dissociation, leaving primarily gp41 immobilized in a trimeric pre-fusion conformation on Ni-NTA ELISA plates (Ringe et al., 2013). Compared to SOSIP.664, IP.664 exhibited much less VRC01 and 2G12 binding, but increased binding of F240 and 7B2 (Figure 1C). These non-neutralizing gp41 epitopes are only exposed when gp120 dissociates. PGT151 was still able to bind IP.664, albeit less efficiently than to intact SOSIP.664 trimers. However, when the I559P mutation, which stabilizes the pre-fusion trimeric conformation of gp41 by impeding transition to the 6-helix bundle post-fusion form (Caffrey et al., 1998; Sanders et al., 2004; Sanders et al., 2002), was also reversed in the WT.664 construct, PGT151 binding was eliminated (Figure 1C). These findings suggest that PGT151 reactivity requires gp41 to be in a trimeric pre-fusion conformation, and that gp120 contributes to the epitope.

Crystal structures of PGT151 and PGT152 Fabs

To characterize the paratope of this class of bnAbs, we solved crystal structures of PGT151 and PGT152 Fabs at resolutions of 1.86 Å and 1.83 Å, respectively (Table 1; Figure 2A and B). Their overall structures are highly similar although we could not compare the heavy-chain complementarity determining region 3 (HCDR3) because 8 residues in the long PGT152 HCDR3 (26 residues) were disordered (Figure 2B). A long HCDR3 loop is a feature common to most other glycan-binding bnAbs (Julien et al., 2013c; Kong et al., 2013; McLellan et al., 2011; Pejchal et al., 2011). Moreover, PGT151 and PGT152 have an unusually long, 16-residue light-chain CDR1 (LCDR1); only 10% of human light chain sequences in the Abysis database (http://www.bioinf.org.uk/abysis/index.html) have an LCDR1 of 16 or more residues.

Figure 2. Crystal structures of PGT151 and PGT152 Fab and use of PGT151 and PGT145 to purify and characterize JR-FL and BG505 EnvΔCT trimers from cell membranes.

Figure 2

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(A) Variable domain from the crystal structure of PGT151 Fab. The HCDR loops 1-3 are labeled and colored in blue, red and magenta, respectively, while the LCDR loops 1-3 are labeled and colored in green, yellow and cyan, respectively. The long 26-residue CDR3 towers above the combining site. (B) Superposition of crystal structures of PGT151 Fab (cyan) and PGT152 Fab (orange). See also Table 1 for data statistics. (C) Scheme for purification of JR-FL EnvΔCT from membranes using PGT151. (D) SEC profile of the purified JR-FL EnvΔCT: PGT151 complex; fractions containing the Fab-trimer complex are labeled in blue. (E) SDS PAGE (reducing and non-reducing) and BN PAGE analysis of the purified JR-FL EnvΔCT: PGT151 complex. (F) Western blot analysis of cell surface expressed BG505 EnvΔCT and JR-FL EnvΔCT trimers, after incubation with PGT151, cell lysis and fractionation on a reducing SDS-PAGE. Gp120 was detected on a western blot to assess the amount of Env captured by PGT151. The cell lysate and protein A flow through (FT) fractions are compared (from step 3 in the purification scheme (C)). Band intensities were analyzed by densitometry in the panels below the gel lanes (absolute intensities: black numbers, relative percentages: blue numbers). Overall, ∼10% of the total gp120 detected (cell surface and intracellular) was captured on protein A as a PGT151-trimer complex, while the remaining ∼90% was in the FT fractions and, hence, non-reactive to PGT151. (G) Blue native PAGE analysis showing the relative stability of JR-FL EnvΔCT or BG505 EnvΔCT complexes with either PGT151 Fab or PGT145 Fab after incubation at 4°C. From left to right: JR-FL EnvΔCT: PGT151 is stable for 40 days (40d) with no dissociation; JR-FL EnvΔCT: PGT145 partly dissociates after 7 days (7d) and is fully dissociated after 40 days (40d); BG505 EnvΔCT: PGT151 is stable for 30 days (30d), whereas BG505 EnvΔCT: PGT145 is partly dissociated. (H) BN-PAGE assessment of the stability of JR-FL EnvΔCT in complex with either PGT151 Fab or PGT145 Fab after incubation at different temperatures for 1h (left and middle). In a densitometric analysis, the intensities of the trimer bands are normalized against the total Env protein content of each lane (right).

PGT151 stabilization facilitates purification of stable, cleaved cell-surface expressed Env trimers

To investigate the PGT151-trimer interaction, we developed a method to extract fully cleaved trimers from the membrane of transiently transfected HEK293F suspension cells using PGT151 IgG. The cleavage-competent Env constructs (JR-FL, BG505, and IAVI C22) contain the gp41 transmembrane helices but lack the cytoplasmic domain and are, therefore, referred to as EnvΔCT. PGT151 IgG-treated cells were lysed with non-ionic detergent and Env-PGT151 complexes were purified on a Protein A column (Figures 2C and 2D). SDS and Blue Native PAGE (BN-PAGE) showed that only cleaved, trimeric Env was purified (Figures 2E and S1A). For all three isolates, only ∼10% of the total Env content (Figure 3F) could be purified in this way, suggesting that only a small proportion of cell surface Env could be extracted in the form of cleaved, native trimers with PGT151-reactive glycans. This proportion is likely larger than 10% on the cell surface, since the method used to quantitate total Env content includes extraneous intracellular, unprocessed Env that is released from within the cell upon lysis.

Figure 3. Membrane-derived, PGT151-purified Env trimers from clades A, B and C all share similar structural features with soluble, cleaved SOSIP trimers and are bound by PGT151 in a sub-stoichiometric manner.

Figure 3

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(A-C) Top (left) and side views (right) of three different EnvΔCT: PGT151 Fab EM reconstructions, showing two Fabs bound per trimer. From left to right, the Env trimers are: (A) BG505 (clade A), (B) JR-FL (clade B), and (C) IAVI C22 (clade C). (D) For comparison, top and side views are shown for the BG505 SOSIP.664: PGT151 Fab complex reconstruction. (E) Comparison of the PGT151-liganded JR-FL EnvΔCT trimer reconstruction (pink (same as B)) with the cryo-electron tomographic reconstruction of the unliganded BaL-1 virion-associated Env spike (EMDB ID: 5019) on the viral surface (gray). See also Figure S4 and Table S1. (F) ITC analysis of high affinity PGT151 binding to BG505 SOSIP.664 trimers. The affinity and stoichiometry were calculated from an average of two experiments. The N=1.3 value is higher than the previously reported 0stoichiometry of N=0.6-0.8 for one PG9 binding to the BG505 SOSIP.664 trimer (Julien et al., 2013b) and lower than the N= 2.3-2.4 for PGT121, PGT128 and 2G12 binding to the same trimer (Sanders et al, 2013). The inference is that ∼2 PGT151 Fabs are bound per trimer. Data analysis was carried out using a single-site binding model. The ITC data do not fit a 2-site model, which argues against a cooperative binding modality. The KD was determined as an average from 3 measurements. Binding affinity was further assessed by bio-layer interferometry in comparison with two other bnAbs (Fig. S1C) (G) Densitometric analysis of a reducing SDS PAGE of JR-FL EnvΔCT trimers purified from cell membranes using either PGT151, PGT128 or PGT145. The black numbers denote the respective peak intensities. The blue numbers in brackets indicate the relative intensity of the respective peak compared to the corresponding peak on the PGT151 panel. While the intensities of the gp120 bands are comparable (0.94-1.0) in each case, the relative intensities of the Fab bands are 0.6 and 1.5 for the PGT145 and PGT128 purifications respectively, indicating that the PGT151 stoichiometry lies between PGT145 (which binds with a stoichiometry of 1 per trimer (McLellan et al, 2011; Sanders et al, 2013; Walker et al, 2011)) and PGT128 (which binds with a stoichiometry of 3 per trimer (Pejchal et al., 2011)). Note that PGT128 only allowed for extraction of a very small amount of Env, which was only sufficient for SDS-PAGE. Neither the yield nor the purity of the sample was in the range of that achieved with PGT151 or PGT145.

We tested PG9, PGT128 and 2G12, but these bnAbs did not enable Env trimers to be purified efficiently, or in large quantities, from the cell membrane. The quaternary-specific bnAb PGT145 (Walker et al., 2011) did facilitate purification of some JR-FL EnvΔCT trimers, but the yield was lower. The PGT145-trimer complexes were also substantially less stable than the corresponding PGT151 complexes, which did not detectably dissociate during multi-week storage at 4°C (Figure 2G). To further characterize the stabilizing effect of PGT151, we incubated the JR-FL EnvΔCT PGT151 and PGT145 complexes for 1 h at 4°C, 37°C, 42°C or 65°C and evaluated the degree of dissociation using BN-PAGE. The PGT145-trimer complex dissociated into dimers and monomers at 42°C, whereas the corresponding PGT151 complex was stable at this temperature (Figure 2H). Both complexes disintegrated completely at 65°C (Figure 2H). Taken together, these data illustrate that quaternary-specific antibodies, particularly PGT151, stabilize the highly labile Env trimer structure.

Membrane-extracted Env trimers from clades A, B and C are structurally similar to soluble SOSIP.664 trimers

EM reconstructions of PGT151 Fabs in complex with EnvΔCT trimers from clades A (BG505), B (JR-FL) and C (IAVI C22) were determined at resolutions of ∼22 Å, ∼19 Å and ∼25 Å, respectively (Figure 4A-C). All three reconstructions were highly similar, with the following correlation coefficients between maps: JRFL: BG505 = 0.97, JRFL: IAVI C22 = 0.96 and BG505: IAVI C22 = 0.96. Whereas the EM reconstructions were refined without imposing symmetry, the three-fold lobes characteristic of the virion-associated Env trimer (Liu et al., 2008) were readily apparent. Two dumbbell-shaped densities indicative of Fabs emanated radially from the trimer. The Env trimer in the EM reconstruction also resembled the unliganded BaL Env tomogram (EMDB ID: 5019), which contained the full gp120 and gp41 ectodomain as well as the transmembrane region (Figure 3E). For all three Env sequences, we did not observe more than 2 PGT151 Fabs bound per trimer. This finding is consistent with densitometric analysis of the SDS PAGE of the purified complexes and the sub-stoichiometric binding between the PGT151 Fab and the BG505 SOSIP.664 trimer in ITC and SEC-MALS (Figures 3F-G and S1B).

Figure 4. The PGT151 epitope on BG505 SOSIP.664 trimer consists of residues from gp141 and gp120.

Figure 4

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(A) PGT151 binding, assessed by Ni-NTA ELISA, is reduced when His6-tagged BG505 SOSIP.664 variants contain certain substitutions in the C5 region of gp120. In the control, 2G12 binding is only mildly affected by the same substitutions. (B) PGT151 binding, assessed by ELISA, is reduced when BG505 SOSIP.664 variants contain certain substitutions in gp41. Point substitutions of N611 and N637 that eliminate glycan sites are shown in the lower panel. For comparison, most substitutions had no effect on 2G12 binding, although the K601A and N607K changes did cause a modest reduction. Assays were done using His- or D7324-tagged BG505 SOSIP proteins (as indicated in the figure). (C) Summary of substitutions in BG505 SOSIP.664 trimers that affect PGT151 binding. Binding ability is scored on a scale from (−, no binding) to (++++, strong binding). The analysis is derived from mean values of 3-5 experiments for each of the variants. Color-code: red: weak to no binding, yellow/orange: moderate binding, green: strong binding. Trimerization and cleavage were comparable to WT SOSIP as assessed by SDS-PAGE and BN-PAGE (data not shown). We cannot exclude the possibility that mutations proximal to the SOS disulfide bond (residues 501 and 605) change the local stability of the trimer and, therefore, binding by PGT151. Both direct and indirect effects are consistent with PGT151 interactions at the gp120-gp41 interface.

The EM reconstructions of membrane-derived BG505 EnvΔCT complexes with PGT151 Fab allowed a direct comparison with PGT151 complexes formed with recombinant, soluble, cleaved BG505 SOSIP.664 gp140 trimers (Figure 3D). The two reconstructions were highly similar overall (correlation coefficient of 0.96), as were the mode and orientation in which the Fab interacts with these two versions of the BG505 trimer (Figures 3B and 3D). Thus, despite the engineered deletion of the MPER and transmembrane regions and introduction of the SOS and I559P trimer-stabilizing changes, the soluble BG505 SOSIP.664 trimers are structurally similar to cell membrane-derived trimers at the moderate resolution obtained here; in particular, both trimer forms contain the highly complex, quaternary and glycan-dependent PGT151 epitope.

In contrast to JR-FL and BG505 EnvΔCT, which both bound two PGT151 Fabs, ∼60% of the IAVI C22 trimers bound only one, as observed by EM. Of note is that PGT151 neutralizes the IAVI C22 virus to a maximum extent of only 60%, whereas JR-FL and BG505 viruses are both neutralized to 100% (Falkowska et al., 2014). These two observations may be linked mechanistically, but understanding this relationship will require further experiments. The BG505 SOSIP.664 trimers also varied in their binding of PGT151. EM studies revealed that approximately 50% of the trimers bound two Fabs while the other 50% only bound one Fab, even when a 6-fold molar excess of Fab was present (data not shown). The underlying basis for this variation with SOSIP trimers remains to be determined but is perhaps rooted in some heterogeneity in glycan composition in the SOSIP trimers, as previously postulated (Lyumkis et al., 2013). Based on our collective observations, PGT151 recognizes intact Env trimers across different clades in a similar manner and binds with a stoichiometry of no greater than two per trimer.

PGT151 binds a quaternary inter- and intra-protomer epitope on SOSIP trimer

From analysis of the EM structure, we designed and screened several BG505 SOSIP.664 trimer variants containing single and double amino-acid substitutions for PGT151 Fab binding by ELISA. Residues from gp120 and gp41 both influence binding to, and formation of, the PGT151 epitope, including several in the gp120 C5 region (K490, T499, R500, R503) and gp41 (K601, N607, N611 and N637) (Figure 4, residues listed conform to HXB2 numbering). However, mutation of T499, R500 and K601 residues had no effect on PGT151 neutralization on the JR-CSF strain (R503S renders the virus inactive and K490 and N607 mutants were not tested), whereas mutation of T499 and neighboring residues did have a weak effect on neutralization in the context of the LAI strain (Falkowska et al., 2014), indicating that these gp120 residues might influence the PGT151 epitope indirectly and/or only in certain Env subtypes. The N611Q and N637Q substitutions (glycosylation sites in wild type) in gp41 resulted in significantly reduced PGT151 binding to BG505 SOSIP.664 trimers, and the double substitution led to a complete loss of PGT151 binding (Figure 4B). The corresponding mutations in the JR-CSF virus also markedly reduced PGT151 neutralization (Falkowska et al., 2014). Hence, these canonical N-linked glycosylation sites, believed to harbor complex glycans (Go et al, 2009), likely comprise part of the PGT151 epitope. The subtle effects (and isolate differences) in both the neutralization (Falkowska et al., 2014) and ELISA assays highlight the difficulty in characterizing complex epitopes. The glycan promiscuity, as seen previously in PGT135, PGT121, and PGT128 binding, introduces further complexity in defining a single epitope ((Kong et al, 2013), Sok, et al, 2014. The direct visualization with the structural data is extremely valuable to define the location, nature and extent of the PGT151 epitope. The exact extent of this epitope may be slightly different between isolates as PGT151 can accommodate some variability and single point mutations (Falkowska, et al., accompanying manuscript).

Fitting the crystal structure of the PGT151 Fab and the EM and x-ray structures of the BG505 SOSIP.664 trimers ((Lyumkis et al, 2013), PDB ID: 3J5M; (Julien et al, 2013a), PDB ID: 4NCO)) into the reconstruction of the JR-FL EnvΔCT: PGT151 Fab complex showed that the epitope involves regions of both gp120 and gp41 (Figure 5A). This conclusion is consistent with the absolute requirement for a fully native trimer conformation (Falkowska et al, 2014). The correct orientation of the Fab in the reconstruction was determined using cytochrome B562RIL (BRIL) engineered into the antibody LC that added extra density to one side of the Fab (Figure S2). In this orientation, the light chain CDR loops are in close proximity to and possibly interact with gp120 glycans N262 and N448 from one protomer, while LFR3 may contact N276 from the adjacent protomer (Figure 5B). The LCDR1 is in close proximity to the C1 region of one gp120 protomer and may contribute weakly to antibody affinity (Figure 5C). The long HCDR3 reaches into the interprotomer cavity at the interface between gp120 and gp41 (Figure 5D-E). In the high-resolution SOSIP.664 x-ray and EM structures (Julien et al., 2013a; Lyumkis et al., 2013), this region contains additional unmodeled density that likely corresponds to the fusion peptide proximal region of HR1 and the fusion peptide. Additionally, the back face of the HCDR loops contacts the adjacent gp41, in a region of HR2 containing glycans at N637 and potentially N611 (Figure 5D). Overall, these data illustrate that PGT151 binds a complex quaternary epitope that involves the gp120-gp41 interface of one protomer and one or more glycans that are attached to the gp41 HR2 region of the adjacent protomer. This epitope does not overlap with any previously identified bnAb epitopes on the Env trimer (Figure 6).

Figure 5. Modeling of the PGT151 epitope in the context of the high-resolution EM structure of the soluble, cleaved BG505 SOSIP.664 trimer.

Figure 5

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(A) Top and side view of the reconstruction of the JR-FL EnvΔCT: PGT151 Fab complex, fitted with the atomic-level EM model of the BG505 SOSIP.664 trimer (PDB ID: 3J5M) (Lyumkis et al., 2013). The gp120 protomers are in gray and the gp41 HR1 and HR2 helices in purple. On the 2 copies of PGT151 Fab, the heavy chain (HC) is in dark blue and the light chain (LC) in light blue. (B) Top view as in (A). The PGT151 LC is predicted to interact with glycans from two gp120 protomers (labeled gp120 A and B), and amino-acid residues from one gp120 protomer. The putative interacting glycans are depicted as spheres in shades of yellow, and the glycosylation site position (Asn) is shown in orange. LCDR coloring is shown as in Figure 2A. (C) The same view as in (B), showing regions of C1 that could potentially interact with LCDR1. In light pink are residues 56-63, and dark pink 75-82. (D) In a side view as in (A), putative PGT151 HC interactions with one gp120 protomer (gray) and two gp41 protomers (purple) are shown. The map shows a single gp120 and the gp41 trimer, segmented from the high-resolution EM structure 3J5M. The HR helices of gp41 B are depicted as purple ribbons. HCDR3 appears to interact with gp140 protomer A at the gp41-gp120 interface, near the fusion peptide proximal region (FPPR) shown in brown. The N637 glycosylation site from protomer B shown in orange is in close proximity to the PGT151 HC. (E) The glycan at residue N637 is modeled as a tetra-antennary GalNac complex glycan that was shown to interact with all members of the PGT151 family (Falkowska et al., 2014). The EM density in cyan, not modeled in the high-resolution structures, is predicted to encompass the putative location of the HR2 residues 611-624 as depicted by the dotted line. The orange circle and arrow respectively indicate the putative position and orientation of N611 and its glycan based on the neutralization assay. See also Figure S2, and movie S1.

Figure 6. PGT151 binds to a new site of vulnerability that does not overlap with any other bnAb epitopes.

Figure 6

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The JR-FL EnvΔCT: PGT151 Fab reconstruction of the new PGT151 epitope compared with the docking of representative Fabs that recognize three of the four previously known bNAb epitope clusters (V1V2 plus N160 glycan, N332 glycan, CD4 binding site). We cannot exclude the possibility that access to the CD4bs might be blocked partly by the two bound PGT151 Fabs; accordingly, PGV04 binding to the CD4bs is shown on the third protomer interface that remains does not have PGT151 bound. The PGT151 binding site on the open face is also shown in blue for reference, with the PGT151 Fab density removed for clarity (right panel). A similar analysis of BG505 SOSIP.664 trimers produces nearly identical results. See also Figures S3 and S4.

Discussion

The weak, non-covalent association between gp41 and gp120 has made structural studies of either soluble or full-length cleaved Env trimers extremely challenging. Furthermore, uncleaved forms of Env are highly prone to adopting non-native conformations, particularly when expressed as soluble proteins but also when present on the cell surface (Pancera and Wyatt, 2005; Ringe et al., 2013). In the context of the BG505 genotype, SOSIP mutations enable the production of soluble, cleaved trimers that are close antigenic (Sanders et al., 2013) and structural (Julien et al., 2013a; Khayat et al., 2013; Lyumkis et al., 2013) mimics of native Env. Here, we show that the PGT151 bnAb not only selectively recognizes cleaved SOSIP trimers, but also stabilizes pre-fusion native Env trimers extracted from the cell surface, thereby preventing dissociation of gp120 from gp41. Thus, it appears that the engineered SOS disulfide bond between gp120 and gp41 and binding of PGT151 to an epitope proximal to that region are different ways to stabilize the gp120-gp41 interaction within trimers. Accordingly, this property of PGT151 creates a new and highly specific method for extracting native trimers for multiple subtypes (roughly 60% of Env sequences, based on the neutralization breadth, Falkowska et al., 2014) from the membranes of Env-expressing cells; the non-native, and probably more abundant, forms of trimer that are also present on the membranes are not recognized by PGT151 and, hence, are eliminated during the purification process. The broadly neutralizing epitope that we describe here also has implications on the mode and mechanism of neutralization by PGT151. Since CD4 is still able to bind the Env-PGT151 complex, PGT151 likely neutralizes the virus by stabilizing the gp41 pre-fusion conformation and the gp120-gp41 interaction, thus precluding CD4- and/or co-receptor-induced conformational changes required for membrane fusion.

Overall the PGT151 epitope is quite complex and requires gp160 cleavage, a properly formed quaternary gp120-gp41 interface, and fully processed gp41 glycans (i.e., complex forms). Accordingly, PGT151-reactivity may represent one of the best ways to identify and evaluate native, functional Env trimers. No known bnAb has such a complex reactivity profile, although various antibodies with trimer preference (PG9) (Julien et al., 2013b; Walker et al., 2009), cleavage preference (VRC06) (Li et al., 2012), and that may involve both gp120 and gp41 moieties (M43) (Zhang et al., 2012) have been reported. It was not possible under any conditions that we tried to generate an Env trimer complex with three Fabs bound. A detailed mechanistic explanation for binding of less than three Fabs per trimer remains to be determined, but we postulate that binding of the first and second Fabs may induce subtle allosteric effects on the Env structure through inter-protomer interactions, rendering the third epitope of the trimer unavailable or not properly configured for PGT151 binding.

The Env-PGT151 complex may also be useful for antigenicity and immunogenicity studies because PGT151 does not block the CD4 binding site, the N332 supersite of vulnerability, or the N160 antigenic site (Falkowska et al., 2014), on at least one, if not all protomers. Similarly, as passive administration of bnAbs can suppress infection and lower viral titers to undetectable levels for the duration of treatment, members of the PGT151 family may also be useful constituents of bnAb combinations that are being considered for antiviral therapy, in view of the non-overlap between this epitope and others (Balazs et al., 2012; Barouch et al., 2013; Horwitz et al., 2013; Klein et al., 2012b). Thus, this described fifth site of vulnerability expands our understanding of the antigenic surface of Env and offers another opportunity for combating HIV-1.

Experimental Procedures

Expression and purification of PGT151, 152, 145, 128 antibodies

IgG and Fab fragments of all antibodies were expressed in HEK293F suspension cells as previously described (Pejchal et al., 2011).

Crystallization and Data collection

Crystallization trials with PGT151 and PGT152 Fab were set up using the IAVI/Joint Center for Structural Genomics (JCSG)/TSRI CrystalMation robotic system (Rigaku), at a concentration of 13 mg/ml in 50 mM Tris-Cl, 150 mM NaCl, pH 7.5. Initial crystals of the PGT151 Fab fragment were obtained in 40% PEG 600, 0.1M phosphate-citrate, pH 4.2. To improve crystallization of PGT152 Fab, a glycan was removed by a light-chain N107K mutation (PGT152ΔGlyc). Crystals for PGT151 and PGT152 Fabs were optimized by microseeding, which improved crystal formation under several conditions in an optimized screen developed at the JCSG (Deller et al., in preparation). Data were collected at APS 23ID-B and at CLS 08ID-1.

Structure determination and refinement

Structures were solved using PHASER (McCoy, 2007) with an unrelated Fab (PDB: 2FX7) as a search model for PGT151 Fab. For the PGT152ΔGlyc Fab, the PGT151 Fab structure was used as search model. Data reduction and scaling was performed using HKL-2000 (Otwinowski and Minor, 1997). COOT (Emsley and Cowtan, 2004) and PHENIX (Adams et al., 2010) were used for model building and refinement. Data collection and refinement statistics are reported in Table 1.

Expression and purification of JR-FL EnvΔCT, BG505 EnvΔCT and IAVI C22 EnvΔCT proteins

HEK 293F suspension cells were transfected with a pSVIIIenv plasmid expressing either JR-FL EnvΔCT E168K (referred to here as JR-FL EnvΔCT, since the E168K mutation is required for the PG9 epitope but does not affect PGT151 binding) or IAVI C22 EnvΔCT. Alternatively, cells were transfected with a phCMVIII plasm id containing a codon-optimized sequence for either BG505-EnvΔCT or JRFL-EnvΔCT under the control of a CMV promoter. In all cases, the cells were co-transfected with a plasm id expressing the Tat transactivator protein to enhance Env expression. For BG505 EnvΔCT and IAVI C22 EnvΔCT expression, the cells were additionally co-transfected with pcDNA3.1 Furin to enhance gp160 cleavage. The cells were harvested after 40 h, washed and resuspended in PBS before incubation with 100 μg/ml of PGT151 IgG (1 h, 4 °C). The cells were then washed to remove excess IgG and resuspended in lysis buffer (PBS, 0.5% Triton X-100, protease inhibitors (Roche)) before cell debris was removed by centrifugation (30,000xg, 30 min). The supernatant was loaded onto a protein A column, followed by extensive washing and subsequent on-column IgG cleavage with Ficin (20 μg/ml) in 50 mM Tris, pH 7.0, 2 mM EDTA, 150 mM NaCl, 0.1 % DDM for 4 h at RT. The Env-PGT151Fab complex was then eluted by gravity flow and analyzed by SEC. The same procedure was used for purification of Env-PGT145 and Env-PGT128 complexes.

Expression and purification of BG505 SOSIP.664

The BG505 SOSIP.664 construct as well as expression and purification were exactly as described previously (Julien et al., 2013a; Lyumkis et al., 2013; Ringe et al., 2013; Sanders et al., 2013). BG505 IP.664 does not carry the SOS gp120-gp41ECTO disulfide bond, and BG505 WT.664 additionally lacks the I559P mutation (used in Figure 1C).

Ni-NTA-capture and D7324-capture ELISAs for BG505 Env proteins

SOSIP trimer ELISAs were performed as described previously (Sanders et al., 2013) using raw supernatants from cells transiently expressing BG505 SOSIP.664-His- or D7324-tagged BG505 Env proteins, except for Figure 1A, where purified Env trimers were used. The percentage of all Env proteins present in trimeric form was always >60% in the supernatants used for ELISA assays.

Isothermal titration calorimetry (ITC)

MicroCal iTC200 and Auto-iTC200 instruments (GE Healthcare) were used to perform ITC measurements as described previously (Julien et al., 2013b). For the PGT151:BG505 SOSIP.664 titration experiment, the Env trimer was deposited in the cell at concentrations between 4.4 and 4.7 μM, and the Fab was in the pipette at concentrations between 79 and 102 μM. In the glycan: PGT151 titration experiments, PGT151 Fab was added to the cell at 100 μM with glycans in the pipette at 4-5 mM (for synthesis of glycans, see Supplemental Experimental Procedures). Data analysis was carried out with Origin 7.0 software using a single-site binding model.

Electron microscopy and sample preparation

Grids for single particle EM were prepared as described previously (Kong et al., 2013). Data for the JR-FL EnvΔCT: PGT151 complex were collected using an FEI Tecnai F20 electron microscope operating at 120 keV coupled with a Gatan US4000 4k × 4k CCD camera at a magnification of 100,000x resulting in a pixel size of 1.09 Å at the specimen plane. All other complexes were imaged on an FEI Tecnai T12 electron microscope operating at 120 keV coupled with a Tietz TemCam-F416 4k × 4k CMOS camera via automated data collection using the LEGINON interface. The imaging magnification was 52,000x with in a pixel size of 2.05 Å at the specimen plane. All data were collected as described previously (Kong et al., 2013) using an electron dose of 25 – 28 e-/Å2 For the random conical tilt (RCT) method with JR-FL EnvΔCT: PGT151, the sample was prepared as before using a carbon-coated C-Flat 400 mesh grid. The data were collected using the Leginon RCT data collection interface (Yoshioka et al., 2007) on a Tecnai F20 electron microscope at 120 keV, with the tilt pairs at 0° and -50°, using a nominal defocus of 1 um. Micrographs were collected on a Gatan 4k × 4k camera at a magnification of 62,000x and pixel size of 1.75 Å.

Image processing

Particle picking and classification was carried out as described previously (Julien et al, 2013b). Template-based particle picking was carried out on the 60 RCT tilt pairs using 7 representative classes from the single particle reference-free class averages. Picks were automatically aligned via Appion and then assessed manually, yielding 3,152 particle tilt pairs, which were subject to reference-free 2D classification using Xmipp Maximum Likelihood Alignment (Scheres et al., 2005a; Scheres et al., 2005b). Five RCT models were generated from these data, each resulting from approximately 200 particle tilt pairs. Two of the models generated that had well-defined trimer and Fab densities were used as the initial model for projection matching refinement with a template stack of 60 images for 90 iterations. The resulting two models were further refined using a bin 4 substack of 5,798 particles from the single particle dataset for 29 iterations after which the two models were nearly identical (Figure S4). Additional data were collected for the single particle analysis and sorted as stated above. One of these models was selected as the initial model for the final raw particle refinement. Models other than the JR-FL Env: PGT151 trimer were refined from independently generated by common lines in the EMAN2 package using reference-free 2D class averages (Tang et al., 2007). No symmetry was applied to any of the projection matching refinements, which were carried out with the EMAN software package (Ludtke et al., 1999). Refinement parameters and final resolution for each EM reconstruction are summarized in Table S1.

Supplementary Material

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Download video file (11MB, mov)

Highlights.

  • PGT151 binds a gp41-gp120 interprotomer epitope on cleaved Env trimers

  • The PGT151 epitope does not overlap with any other HIV-1 Env epitopes

  • PGT151 enables isolation of functional cleaved Env from the cell membrane

  • Membrane-extracted and soluble SOSIP.664 Env trimers are structurally similar

Acknowledgments

We thank Y.Hua, A. Irimia, L. Kong, H. Tien, M. Deller and M. Hong for technical assistance and valuable discussions and J. Voss for providing expression plasmids. This work was supported by the International AIDS Vaccine Initiative Neutralizing Antibody Center, Scripps CHAVI-ID (UM1 AI100663), NIH grants HIVRAD P01 AI082362 and R01 AI084817, the University of California, San Diego Center for AIDS Research (CFAR), an NIH-funded program (P30 AI036214) (which is supported by the following NIH Institutes and Centers: NIAID, NCI, NIMH, NIDA, NICHD, NHLBI, NIA), Aids Fonds Netherlands grant #2011032, a Vidi grant from the Netherlands Organization for Scientific Research (R.W.S.), a Starting Investigator Grant from the European Research Council (R.W.S.), a German Academic Exchange Service (DAAD) Research fellowship (C.B.), and a Dissertation Fellowship from the California HIV/AIDS Research Program (J.H.L). This study is made possible by the generous support of the American people through the United States Agency for International Development (USAID). The contents are the responsibility of the International AIDS Vaccine Initiative and do not necessarily reflect the views of USAID or the United States Government. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Research described in this paper was performed using beamline 08ID-1 at the Canadian Light Source, which is supported by the Natural Sciences and Engineering Research Council of Canada, the National Research Council Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan. The EM work was conducted at the National Resource for Automated Molecular Microscopy at The Scripps Research Institute, which is supported by the Biomedical Technology Research Center program (GM103310) of the National Institute of General Medical Sciences. Images were generated using the UCSF Chimera package. This is manuscript 26044 from The Scripps Research Institute.

Footnotes

Accession Numbers: Crystallographic coordinates and structure factors are deposited in the Protein Data Bank (PDB) with the following PDB codes: 4NUG (Fab PGT151) and 4NUJ (PGT152Δgly-Fab) and released upon publication. The EM reconstructions have been deposited in the Electron Microscopy Data Bank under the following accession codes (EMD-5918 EMD-5919 EMD-5920 EMD-5921).

Supplemental Data: Supplemental Data includes four Figures, two Tables, one movie and Supplemental Experimental Procedures and can be found with this article online at: http://dx.doi.org/10.1016/j.immuni.2013.XXX

First Author Contributions: C.B. conducted the x-ray crystallographic studies, biophysical characterizations, and Env purification. J.H.L. conducted the electron microscopy experiments and structural modeling. Both C.B. and J.H.L. conducted data analyses and interpretation, and contributed to the writing of this manuscript.

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Contributor Information

Ian A. Wilson, Email: wilson@scripps.edu.

Andrew B. Ward, Email: abward@scripps.edu.

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