Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans

Abstract

New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1). Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.

Figure 1. Structure and sequence characterization of antibodies of the PGT121 family.

A) Superimposition of the PGT121 (green), PGT122 (cyan) and PGT123 (magenta) Fab crystal structures determined at 2.8 Å, 1.8 Å and 2.5 Å resolutions, respectively. Only the variable domains are shown with secondary structure rendering. The three CDRs of the light and heavy chains are labeled and colored in different shades of blue and red to orange, respectively. The 24-residue HCDR3 loop divides the paratope into two faces: an open face composed from HCDR1, HCDR2 and the base of HCDR3 and a more elongated face from LCDR1, LCDR3 and the tip of HCDR3. B) Sequence conservation between antibodies of the PGT121 family mapped on the PGT122 crystal structure, which is rendered as a surface representation. Identical residues in the three antibodies are colored green while divergent sequences are represented in white. Two regions of clustered sequence identity are predicted to play an important role for antigen recognition (conserved faces 1 and 2). These figures were generated using UCSF Chimera . C) Sequence alignment among antibodies of the PGT121 family with the consensus sequence shown above and non-conserved residues shown below for each antibody. The six light chain (LC) and heavy chain (HC) CDRs and Framework Regions (FR) are indicated based on Kabat alignment. This figure was generated using Jalview .

Figure 2. Alanine-scanning mutagenesis of the PGT121, PGT122 and PGT123 paratopes.

A) Surface residues predicted to play a role in mediating antigen recognition were identified from the crystal structures, subsequently mutated to alanine, and the resulting IgG mutants were tested for their ability to neutralize HIV-1 JR-CSF pseudoviruses. The fold increase in the neutralization IC₅₀ compared to WT IgG is reported in the table by color code: green, <2; yellow, 2–5; orange, 5–9; and red, >9. Mapping these results on the PGT121, PGT122 and PGT123 crystal structures allowed identification of a region crucial for mediating neutralization near the elongated face, whereas side chains of residues in the open face appear to play a more secondary role in HIV neutralization. Residues that show an increase in the neutralization IC₅₀ of >9 (red) compared to WT IgG upon mutation to alanine are labeled on the structures. B) The PGT121 open face accommodates a biantennary glycan from a symmetry-related Fab molecule in the crystal (Figs. S2, S3). PGT121 is rendered as a gray surface, whereas the glycan is shown as magenta sticks. The blue mesh is the 2Fo-Fc electron density map countered at a 1.2 sigma level around the glycan moiety. The figure was generated using Pymol.

Figure 3. Negative stain electron microscopy reconstruction of HIV SOSIP.664 trimer in complex with Fab PGT122 and in comparison to PGT128

. A) Top and side views of the BG505 SOSIP.664:PGT122 Fab reconstruction at 15 Å resolution with the fitted crystal structures of PGT122 Fab and gp120 core (PDB ID 3DDN [35]) rendered as secondary structure cartoons. The PGT122 Fab is shown in blue (heavy chain) and white (light chain), and the gp120 core is shown in red. B) Top and side views of the BG505 SOSIP.664:PGT122 Fab reconstruction at 15 Å resolution (cyan) and the KNH1144 SOSIP.664:PGT128 Fab reconstruction at 14 Å resolution (light green), with the fitted crystal structure of PGT128:eODmV3 (PDB ID 3TYG [29]) shown as a secondary structure cartoon. The PGT128 Fab is shown in blue (heavy chain) and white (light chain), and the engineered gp120 outer domain (eODmV3) is shown in red. The proposed locations of V1/V2 loops and the V3 loop in the Env trimer, as well as the location of the CD4 binding site, have been labeled. The figure was generated using UCSF Chimera .

Figure 4. Antibodies of the PGT121 family compete with sCD4, but not PGV04, for binding to gp120 in solution and on the cell-surface.

A. Fold change in binding affinity (K_d) for sCD4 and PGV04 interactions with gp120 when antibodies of the PGT121 family are first pre-complexed with gp120. The order from left to right is as on the inset legend from top to bottom. Negative values indicate a decrease in binding affinity of sCD4 or PGV04 in the presence of PGT121 antibodies, positive values represent an increase in binding affinity and a value of ∼1 represents no effective change in binding affinity. Sequential ITC binding experiments show that sCD4 has over 100 to 650 fold decrease in binding affinity to gp120 when antibodies of the PGT121 family are pre-complexed with gp120. On the other hand, pre-complexing PGT121 antibodies with gp120 does not lead to a change in binding affinity for PGV04 to gp120. These results suggest that antibodies of the PGT121 family do not sterically block access to the CD4 binding site (Fig. S8A), but disturb CD4 binding, possibly by interfering with gp120 conformational changes associated with CD4 binding. No sCD4 competition by PGT121 antibodies is observed for a core-miniV3 gp120 construct, indicating that modulatory elements of gp120 such as C1, V1/V2 and fully length V3 are important in modulating sCD4 competition. B. SEC-purified complexes of gp120 with various Fabs were tested for their ability to bind CD4+ TZM-bl cells by FACS. 17b+gp120 (black) and 2G12+gp120 (orange) complexes bound well to CD4+ TZM-bl cells. On the other hand, a PGT123+gp120 complex (red) was not able to engage CD4+ TZM-bl cells. Lack of binding of the PGT123+gp120 complex to CD4+ TZM-bl cells is comparable to similar lack of binding of gp120 in complex with the CD4-binding site antibody PGV04 (blue). A similar inability to bind CD4+ TZM-bl cells was observed for gp120 in complex with PGT121 and PGT122 antibodies (Fig. S9). PE-A represents the relative intensity of detected Fab on the surface of CD4+ TZM-bl cells. Curves with filled areas indicate Fab alone (negative control), whereas curves with hollow areas are for the Fab-gp120 complexes. C) sCD4 and D) PGV04 binding to cells expressing JRFL Env on their surface as observed by flow cytometry. PGT123 Fab (pink), b12 Fab (yellow) and 17b Fab (green) were pre-incubated with the cells in titrating amounts at 37°C before being exposed to a constant amount of either C) sCD4 or D) PGV04. As expected, b12 Fab that targets the CD4 binding site directly competes with CD4 binding, and 17b Fab, which binds to the co-receptor binding site, enhances CD4 binding. PGT123 Fab competes with sCD4 binding to the same extent as b12 Fab. A similar level of sCD4 competition was observed for PGT121 and PGT122 antibodies (Fig. S9). On the other hand, PGT123 Fab does not compete with PGV04, a CD4 binding site targeted antibody that does not induce conformational changes upon binding . Binding curves are represented by plotting the dimensionless mean fluorescence intensity (MFI) of C) sCD4 and D) PGV04 binding as a function of Fab concentration.

Figure 5. Model of HIV Env recognition by antibodies of the PGT121 family.

A model of the PGT 121 family interaction with Env trimer is shown on the bottom right and was generated from the electron microscopy reconstruction of the unliganded membrane-anchored HIV-1 Env trimer (gray surface, EMDB ID 5019 and 5021 [35]) with modeled glycans and PGT121 Fab as blue spheres and green surface, respectively. The large inset is a close-up view of the fitting of the PGT121 Fab crystal structure (light and heavy chains are colored in light and dark green, respectively) and the eODmV3 crystal structure (colored in gray), as bound by the PGT128 Fab (PDB ID 3TYG [29]) in the negative-stain EM reconstruction (transparent gray mesh). The crystal structures of the PGT121 Fab and the gp120 outer domain are rendered as secondary structure cartoons. PGT121 paratope residues identified as most important for mediating HIV-1 neutralization are shown as red and orange spheres according to the scheme used in Fig. 2. In this model, crucial residues of the PGT121 paratope in the elongated face (rendered as red spheres) are located near the base of the gp120 V3 loop (black). Consistent with the biochemical data, the antibody paratope is located near the N332 glycan (rendered as yellow sticks and surface). Glycosylation at this position is crucial for recognition by antibodies of the PGT121 family. In addition, the PGT121 paratope is located in close proximity to the N301 glycan (rendered as cyan sticks and surface), as well as putative complex glycan (rendered as magenta sticks and surface) observed in the PGT121 crystal structure of unknown location on gp120 but close to V1/V2 in the model. For this complex glycan, the two N-acetylglucosamines (NAG) that would be attached to the Asn on the protein point in the direction of the region associated with gp120 V1/V2 loops. Together, our data show that PGT 121 binding to this epitope appears to allosterically block CD4 engagement. The CD4 binding site is colored blue. The figure was generated using UCSF Chimera .