A Broadly Neutralizing Antibody Targets the Dynamic HIV Envelope Trimer Apex via a Long, Rigidified, and Anionic β-Hairpin Structure.

PGT145 Recognizes Oligomannose Glycans

The glycoforms to which apex antibodies bind have been a matter of debate. Studies have shown that apex antibodies such as PG16 bind sialylated hybrid glycans with much higher affinity than oligomannose glycans (Andrabi et al., 2015, Pancera et al., 2013). Similarly, PG9 showed increased binding affinity, neutralization potency, and maximum percentage neutralization (MPN) in the presence of complex/hybrid glycans (Figure 2A). PGT145-family antibodies have also been shown to preferentially bind to wild-type viruses and trimers compared to Kif treated Envs (Sok et al., 2014). However, SPR analysis of PGT145 Fab binding to BG505 SOSIP.664 trimers demonstrated that PGT145 has the highest affinity for glucose N-acetyltransferase 1 (GnT1)-deficient 293S-produced trimers that only have oligomannose Man_5-9 glycans, although the larger Man_8-9 glycans in the +Kif condition are somewhat inhibitory (Figures 2B, S3B and S3C). Further, a 31-pseudovirus neutralization panel showed a ∼3.5-fold improved median IC₅₀ of PGT145 in 293S compared to wild-type glycan producing 293T cell produced pseudoviruses (Figure 2A), suggesting that, while PGT145 can accommodate glycan heterogeneity, there is a preference for oligomannose forms.

 

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Figure 2

PGT145 Favorably Binds Oligomannose Glycans

(A) IC₅₀ and maximum percentage neutralization (MPN) values for PGT145 and PG9 neutralization of pseudoviruses produced in 293T or 293S cells. ND: Not determined.

(B) A comparison of kinetic parameters derived from SPR analyses of PGT145 binding to BG505 SOSIP.664 trimers produced in trimers expressing different glycoforms and N-linked glycosylation site mutations. Also see Figures S3B and S3C.

Previously, glycan arrays failed to detect PGT145 binding across a wide range of glycans (Andrabi et al., 2015). Here, we employed a dendrimer format of glycan array slides (Wang et al., 2008) that creates a higher density of glycans. PGT145 and PGDM1400 bound the C-isomer of a Man₈ glycan (M8C) (Figures S3D–S3F), but not the Man₈ B-isomer (M8B) indicating that the terminal Man(α1-2) residue of the D3 arm may sterically clash with PGT145-family Fabs. These data suggested that not only the correct glycoform, but also close spacing of glycans is essential for PGT145 binding.

The PGT145 Epitope Consists of Glycans from Two Protomers and Peptide Contacts from all Three Protomers

In the cryoEM map, the primary interacting N160 glycan appeared as oligomannose, consistent with our glycan array analysis. None of the asymmetric N160 glycans revealed density corresponding to core fucosylation (Figures S4A and S4B), often found in complex glycans. The total buried surface area between PGT145 and the modeled N160 glycans was ∼555 Å² (glycan 1: 384 Å², glycan 2: 131 Å², glycan 3: 40 Å²). PGT145 most prominently interacted with a single N160 glycan (N160_glycan1), less extensively with a second N160 glycan (N160_glycan2), and likely not at all with the third N160 glycan (N160_glycan3) (Figures 3A–3D, S4A–S4C). The density resolved for N160_glycan1 is approximately the size of Man₆ (Figure S4A) and its core GlcNAc sugars bound the peptide backbone of the HCDR3 descending strand, while the D1 arm packed between HCDR2 and HCDR3, making polar contacts with highly conserved H52a_HCDR2 (Figure 3B). Y100f_HCDR3—a tyrosine known to be sulfated—orients R100a _HCDR3, and the sulfate might also interact with N160_glycan1 Asn, while the benzene ring face of Y100f _HCDR3 is packed against T162 (Figure 2B). Although the D2 and D3 arms of N160_glycan1 are not fully resolved, we infer from glycan array data that the terminal D3 arm would clash with the LC of PGT145 in its orientation in the structure (Figures 3A and S3E). Together, the data predicted that much of the glycan recognition is driven by interactions made with the GlcNAc stalk, with steric accommodation for a Man₆, Man₇, Man₈, or even a hybrid glycan at N160_glycan1. In contrast, a complex glycan with its bulkier Gal(β1-4)GlcNAc(β1-2/4/6) branching units would likely clash with PGT145. Thus, the mode of N160 glycan recognition differed from that of the PG9-like antibodies, in which the N160 glycan branches bury into a pocket formed by the hammerhead-shaped HCDR3 and the LCDRs (Figure S4D). The GlcNAc base of N160_glycan2 interacted with Y100c_HCDR3 and to some extent with LCDR1, although not well resolved in our structure (Figure 3C). LCDR1 is the most variable CDR segment among the PGT145-family variants (Figure S1B), suggesting that the PGT145-family antibodies might accommodate N160_glycan2 in different ways. N160_glycan3 projected away from the antibody likely because it is sterically inhibitory to PGT145 binding in its native conformation (Figures 3D, S4C). Consequently, the N160 glycan is both essential (on one, probably two protomers), as well as inhibitory (on the third protomer) to binding of PGT145, presenting a unique challenge to epitope recognition of an asymmetric antibody molecule at a symmetry axis.

 

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Figure 3

Simultaneous Engagement of Glycans and Protein Residues from Multiple gp120 Protomers Is Required for PGT145-Class bnAb Binding

(A) PGT145 interaction with N160_glycan1. Glycans are shown as green sticks, and the CDRs are colored as in Figure 1A. The arrow indicates the direction of the D3 branch of the glycan.

(B) N160_glycan1 interacts with the PGT145 HCDR3 peptide backbone. The red star indicates a sulfated tyrosine.

(C) PGT145 interaction with N160_glycan2.

(D) PGT145 interaction with N160_glycan3.

(E) Neutralization IC₅₀ values of 293T cell-produced BG505 pseudovirus mutants by PGT145-family variants.

(F) Left and right panels: R166 and K121 from all three gp120 protomers (white) interact with the electronegative moieties in HCDR3. Red star indicates tyrosine sulfation observed in the X-ray structure of the Fab.

(G) Residues in HCDR3 along with K169_gp120 support interactions with the base of N160_glycan2.

In contrast to hammerhead HCDR3-type apex antibodies, which are dependent on the N156 glycan and bind complex glycoforms on glycan arrays (Andrabi et al., 2015, Gorman et al., 2016, McLellan et al., 2011, Pancera et al., 2013), none of the N156 glycans directly contacted PGT145 (Figures S4D and S4E). Ablation of the N156 glycosylation site by an N156K substitution abrogated neutralization by PGT145 (Figure 3E). We therefore looked for an indirect effect that could be ascribed to differential processing of N160 glycans in N156 glycan-lacking Env variants expressed in different cell-lines. SPR showed negligible binding of PGT145 Fab to all of the N156D substitution variants (Figure S3B), and inspection of the N156D mutant by negative stain EM revealed that this mutation enriched for non-native-like trimers (Figure S4F). Thus, the reduced binding of PGT145 to N156 glycan knockouts most likely arose from disruption of the conformational epitope through the loss of the inter-V2-strand interaction between Y173 and N156 core GlcNAc. Residue 173 is a Tyr, His, or Phe in >86% of Envs, all of which are suitable for stabilizing the N156 glycan. Indirect glycan effects on the PGT145 epitope might also result from variability in the length and number of glycosylation sites on V1 and V2 loops, as such viral attributes have been noted to increase with disease progression and correlate with reduced neutralization sensitivity (Curlin et al., 2010, Sagar et al., 2006). The most variable segment of V2, which is distal to the 3-fold axis, is flexible and disordered in the majority of high-resolution SOSIP.664 structures. The density corresponding to the projection of the V2 loop in BG505, however, suggested that its glycans would be close to the bound PGT145 Fab, and the N-linked glycosylation sites at N185e and N185h could restrict accessibility to the epitope (Figure S4G). Removal of the N185h glycan site in particular, led to a ∼1.5-fold increase in the association constant of PGT145 Fab, and an increase in the stoichiometry (S_m) from 0.87 to 0.96 (Figures 2B, S3B and S3C), confirming that outer-V2 region glycans can partially shield the apex bnAb epitope.

Peptide contacts between PGT145 and Env (total buried surface area ∼729 Å²) are largely restricted to interactions between the tip of HCDR3 with the C-strand of V1/V2 and the C1 region near the base of V1 including K121 (Figure 3F). K121 from each protomer interacted with sulfated tyrosine Y100i, while R166 from each protomer interacted with various electronegative moieties in HCDR3 of PGT145 (Figure 3F). Finally, Y100m_HCDR3 stacked up against the hydrocarbon segment of K169, which probably helps engage the N160_glycan2 via a network of interactions along with E100o (Figure 3G).

To confirm our structural observations, we conducted alanine-scanning neutralization assays on BG505 pseudovirus mutants (Figures 3E, S5A). Neutralization was reduced when the N160 or N156 glycan sites were knocked out (N156K, N160K, N160A, T162A), or direct peptide contact residues were substituted (K121A, R166A). Point mutations in V1/V2 that caused BG505 SOSIP.664 trimers to adopt aberrant trimer forms (M161A, L165A, D167A) when observed by EM, selectively decreased neutralization potencies of PGT145-family bnAbs versus hammerhead-group, PG9 and CH01 (Figures 3E, S5). Neutralization by CAP256-VRC26.09 was affected mostly by the same Env mutations that affected the PGT145 class. Thus, despite CAP256-VRC26.09 having a HCDR3 structure that more closely resembles the hammerhead-class (Doria-Rose et al., 2014), it is dependent on proper quaternary apex formation like PGT145, and engages all three protomers upon binding. Overall, our results further classified PGT145 as a distinct class of apex bnAb, compared to PG9, and that it is an N160 glycan-dependent antibody that relies on simultaneous recognition of peptide contacts from the apex central residues of all three gp120 V1V2 loops, and is indirectly affected by the glycan at N156.

PGT145 Recognizes an Electropositive Sink at the Trimer Apex

Apex-bnAbs recognize the conserved electropositive V2, particularly strand C (Doria-Rose et al., 2012). We looked for additional residues near the central electropositive layer that could add to the overall local charge. K117, which is 97% conserved, is deeply buried under the trimer apex and is likely important for PGT145 recognition. Additionally, 84% of clade B Envs have an arginine or lysine at V3 position 315, instead of glutamine commonly found in other clades, effectively increasing the positive charge potential at the center of the trimer apex (Figure S6). Residue 315 is located between the electropositive layer created by strand C and K121, which is part of the PGT145 epitope, and thus the additional electropositive layer might interact favorably with the electronegative HCDR3 (Figure S6E). However, PGDM1400- and CAP256-VRC26-lineage bnAbs have lower clade B breadth (Doria-Rose et al., 2015, Sok et al., 2014). Therefore, this extra positive residue likely influences trimer apex dynamics through electrostatic repulsion, as often observed in clade B SOSIP trimers (de Taeye et al., 2015, Pugach et al., 2015). An increased tendency to “breathe” and open up at the apex would both alter the apex epitope but also enhance accessibility to the CD4bs, consistent with clade B Env infected-individuals preferentially generating CD4bs bnAb responses, but reduced apex responses (Rusert et al., 2016).

To determine whether there is any correlation between PGT145 IC₅₀ and electropositive charges near the PGT145 epitope, we analyzed sequence variability at select positions in relation to neutralization using a linear regression model (Figure 4A). The goal of the model was to weigh the contribution of each residue to the escape phenotype defined by two different IC₅₀ cut-off values, by considering the amino acid variation in each strain and their respective neutralization IC₅₀ values. This weighting was reflected in the magnitude of the regression coefficient. The model predicts that the larger the magnitude of this coefficient, the more the residue influences resistance. In this analysis, residues that contact PGT145 and are important for neutralization, such as K121, R166, and T162, are also well conserved, having an exact match conservation score (relative to BG505) of ∼70% or higher (Figure 4A). This analysis also revealed that K171 is important even though it does not directly contact PGT145. K171 seemed to interact with the base of the N160 glycan and/or restrict the movement range of N156 and N160 glycans to indirectly affect PGT145 binding (Figures 3G and ​and4B).4B). A subtler effect on PGT145 neutralization was observed among hydrophobic residues involved in stabilization of the trimer apex (Figures 4A, 4C, and 4D). Particular V1/V2 residues have neutralization regression coefficients that were more prominent with an IC₅₀ > 1 μg/mL cut-off, such as L125, I309, L175, and I326, which also tend to be more conserved. In binding, the anionic HCDR3 of PGT145 stabilizes the trimer by offsetting the net charge at the apex, especially when Env contains additional cationic residues. Mechanistically, Env requires CD4 triggering to transition into an open state leading to exposure of the co-receptor binding site. The proximal localization of positively charged residues results in repulsion between the gp120 protomers, contributing to meta-stability and priming Env for CD4 induced conformational changes. Too much repulsion between protomers would result in a constitutively open trimer susceptible to degradation and therefore render the virus defunct. Thus, we propose that a fine balance between tight hydrophobic packing and localization of electropositive charge is required for viral fitness, which PGT145 exploits for epitope targeting (Figure 4E).

 

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Figure 4

PGT145 IC₅₀ Correlates with gp120 Residues Involved in Trimer Stability

(A) Logistic regression analysis was used to model neutralization escape based on amino acid sequence information. An IC₅₀ of > 10 μg/mL (top) or > 1 μg/mL (bottom) was used to define escape. The relative conservation of the given residue is shown as a line graph. Residue-161 (asterisk) is also part of the direct PGT145 epitope.

(B) Y173 and K171 interact with N156 and N160 to stabilize V1/V2 and restrict glycan conformations.

(C) Hydrophobic packing interactions in hydrophobic core 1.

(D) Stabilizing interactions in hydrophobic core 2.

(E) Env is a meta-stable complex that transitions from a closed (top center and right) to an open unliganded trimer (top left) configuration. CD4 triggers a transition to the CD4-bound configuration (left) with an exposed co-receptor binding site (purple patch). The electronegative paratope of PGT145 (red patch, lower right) recognizes an electropositive pocket (blue), which is only present on a stable trimer. Both viral fitness and PGT145 reactivity require balance between electropositivity (blue) and hydrophobicity (tan) at the trimer apex (bottom left graph).

Negative Stain EM Data Collection and Processing

All negative stain grids were prepared using 400 Cu mesh carbon coated grids as previously described (Julien et al., 2013b). PGT145 bound complexes were stained using NanoW for ∼30 s, and unliganded trimers were stained using 2% uranyl formate for 45 s to 1 min. The EnvΔCT-PGT145 Fab complex was imaged on a Tecnai T12 coupled with a Tietz PXL 2k × 2k CCD camera, at a magnification of 52,000× resulting in 2.65 Å/pix images. Images were collected using Leginon (Suloway et al., 2005). Particles were processed as previously described (Blattner et al., 2014). Unliganded trimer mutants were imaged on a Tecnai T12 microscope coupled with a Tietz TemCam F416 CMOS detector, at a magnification of 52,000× resulting in 2.05 Å/pix on the specimen plane. Reference-free 2D classes were generated using MSA/MRA (Ogura et al., 2003) to sort the different trimer forms. Because these trimers were SEC purified over a Superdex 200 Increase 10/300 GL column, a large number of the mutants contained a significant portion of particles corresponding to monomers and dimers. Thus monomer and dimer populations were included in the analysis. In the first round of classification, all classes that had more than one particle in the boxed class average were eliminated. From a second round of 2D classification, reference-free 2D class averages were sub-grouped into three populations; (1) “non-native” that includes monomers, dimers, and badly assembled trimers, (2) “native-like closed,” and (3) “native-like total” that includes both the closed (population [2]) and “breathing” (“native-like” but open) trimers (Pugach et al., 2015) (Supplemental Experimental Procedures).

CryoEM Data Collection and Processing

BG505 SOSIP.664 trimers produced in 293F cells were pre-complexed with 6-molar excess of 3BNC117 Fab overnight at 4°C, and size exclusion purified. To break the pseudo-symmetry of PGT145, PGT145 Fab was pre-complexed with a mouse Fab obtained from a commercial hybridoma cell line ATCC CRL-1757 (1757) that binds the HC of human Fabs (Figures S2A and S2B). To make the BG505 SOSIP.664-3BNC117-PGT145-1757 complex, PGT145 was combined with 1757 Fab (1:2 molar ratio) and size exclusion purified. The PGT145-1757 complex was then incubated with the purified BG505 SOSIP-3BNC117 complex and size exclusion purified. Samples were concentrated and manually frozen in liquid ethane. Data were collected via Leginon (Suloway et al., 2005) on an FEI Titan Krios electron microscope operating at 300 KeV coupled with a K2 Summit direct electron detector camera (Gatan) in counting mode at a magnification of 22,500× resulting in a pixel size of 1.31 Å/pixel, using a total dose of ∼32 e⁻/Ų. Data were processed using RELION 1.4b1 (Scheres, 2012). The BG505 SOSIP.664-3BNC117 complex was refined using 22,625 particles with C3 symmetry imposed, to ∼4.4 Å resolution at a Fourier shell correlation (FSC) cut-off of 0.143. 1757 was found to be specific for the constant region of the Fab HC, near the protein G binding site (Derrick and Wigley, 1994) (Figure S2F) allowing for confidence in the PGT145 Fab orientation. The BG505 SOSIP.664-3BNC117-PGT145-1757 and BG505 SOSIP.664-3BNC117-PGT145 classes from 3D sorting were combined, resulting in a total of 65,060 particles that were refined without imposing symmetry to 4.7 Å resolution (FSC = 0.143). A soft edge mask masking out 1757 Fab and PGT145 Fab constant domains was applied for one additional iteration of focused refinement, resulting in the ∼4.3 Å resolution model (FSC = 0.143) (Figure S2D) (Supplemental Experimental Procedures).

Model Building and Refinement into the CryoEM Maps

The crystal structures of BG505 SOSIP.664 (4TVP) and 3BNC117 Fab (4JPV) were used as templates to generate an initial atomic model using the Modeller plug-in in UCSF Chimera (Pettersen et al., 2004, Webb and Sali, 2016). The resulting model was iteratively fixed and refined in Coot (Emsley et al., 2010) and RosettaRelax (DiMaio et al., 2009) employing Ramachandran constraints. Final models were chosen based on a combination of the Rosetta energy score, MolProbity and clash scores (Chen et al., 2010), and EMRinger score (Barad et al., 2015). Glycans were modeled into the finalized protein model as previously described (Lee et al., 2015), with all glycans being modeled as oligomannose. The protein structure in the BG505-3BNC117 model was used as an initial model to refine the PGT145-bound structure. The sulfated tyrosines in the PGT145 Fab X-ray structure were replaced with regular tyrosines because Rosetta fails to recognize sulfated tyrosines. The complete BG505-3BNC117-PGT145 complex was refined and followed by glycan modeling as was done for the BG505-3BNC117 complex (Supplemental Experimental Procedures).

Crystallization and X-ray Data Collection

PGT143 and PGT144 Fabs were concentrated to 4-24 mg/mL. Fab samples were screened for crystallization using the 384 conditions of the JCSG Core Suite at both 277 and 293 K using the TSRI/IAVI/JCSG robotic Crystalmation system as described previously (McLellan et al., 2011). Data collection was performed at cryogenic temperature (100 K) at beamline 23-ID of the Argonne Photon Source (APS), using a beam wavelength of 1.033 Å. The diffraction data were indexed, processed and scaled with HKL-2000 (Otwinowski and Minor, 1997) or XDS. Both structures were determined by molecular replacement using Phaser (McCoy et al., 2007) with PGT145 Fab as an initial model (3U1S). Model building and refinement was carried out using Coot-0.7 (Emsley et al., 2010) and Phenix (Adams et al., 2010) (Supplemental Experimental Procedures).

Surface Plasmon Resonance

SPR analysis of PGT145 Fab binding to His-tagged BG505 SOSIP trimers was analyzed on a Biacore 3000 instrument at 25°C. Glycan knockout mutants were expressed in HEK293F cells unless otherwise indicated. All trimers were immobilized on the chip by His-tag capture, as previously described (Yasmeen et al., 2014). To study interference or enhancement of CD4 and PGT145 binding, sequential binding analyses were also performed as stated previously (Derking et al., 2015) (Supplemental Experimental Procedures).

Neutralization Assays

For pseudovirus production, we cotransfected HEK293T or 293S cells with an Env encoding and an Env-deficient backbone (pSG3DEnv) plasmids using Fugene 6 (1:2 ratio). Pseudoviruses were harvested 48–72 hr post-transfection, filtered, and titrated for use in neutralization assays. Neutralization was measured in TZM-bl target cells, as described previously (Andrabi et al., 2015) (Supplemental Experimental Procedures).

Glycan Array Assays

mAbs were screened on a custom high mannose array, consisting of 9 mannosides and 1 control sialylated N-glycan. The 10 amine-linked glycans were covalently immobilized onto custom NHS-ester dendron functionalized glass microscope slides (G3 and G4, ZBiotech). To assess mAb binding, the antibodies were pre-mixed with the detection antibody (anti-human-IgG R-PE). Following 15 min, the pre-complexed antibodies were applied directly to the slide surface and allowed to incubate for 1 hr and then washed. Washed arrays were dried by centrifugation and then scanned for RPE signal on a confocal microarray scanner (Supplemental Experimental Procedures).

This work was supported by the California HIV/AIDS Research Program Dissertation Award D12-SRI-353 (to J.H.L.), the Scripps CHAVI-ID (UM1 AI100663), P01 AI110657, International AIDS Vaccine Initiative, and the Bill and Melinda Gates Foundation CAVD (OPP1115782 and OPP1084519). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This is manuscript number 29440 from the Scripps Research Institute.