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. 2010 Nov 18;6(11):e1001195.
doi: 10.1371/journal.ppat.1001195.

Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41

Charles Sabin  1 , Davide Corti, Victor Buzon, Mike S Seaman, David Lutje Hulsik, Andreas Hinz, Fabrizia Vanzetta, Gloria Agatic, Chiara Silacci, Lara Mainetti, Gabriella Scarlatti, Federica Sallusto, Robin Weiss, Antonio Lanzavecchia, Winfried Weissenhorn
Affiliations

Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41

Charles Sabin et al. PLoS Pathog. .

Abstract

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HK20 Fab binds to a conserved epitope on gp41 HR1.
(A) Ribbon diagram of the HK20 Fab (heavy chain in blue and light chain in green) approaching its epitope on gp41 (HR1 in yellow and HR2 in grey) looking down the three-fold axis of gp41 (upper panel). The lower panel shows the side view of the complex and shows that the Fab complex approaches 5-Helix with an approximate angle of 60°. (B) Close-up of the HK20 interaction with 5-Helix. The complementarity determining regions are coloured differently and labelled H (heavy chain) and L (light chain).
Figure 2
Figure 2. Close-up of the HK20-5-Helix interactions.
(A) Only CDR H2 (blue), H3 (green) and L3 (yellow) contact two HR1 helices of 5-Helix. (B) Close-up of the hydrophobic and polar interactions mediated by CDR H2. (C) Close up of CDR H3 interactions highlighting the sandwich structure produced by residues H564, Y97, W571, P100b and Y100c. (D) Close-up of the polar network formed by CDR H2 N58, H3 Y100c and gp41 HR1 chain A Q575.
Figure 3
Figure 3. The gp41 prehairpin structure can bind three HK20 mAbs simultaneously.
Molecular model demonstrating the docking of three HK20 Fabs onto three HR epitopes formed by three HR1 helices (Figure 1A; a-c, c-b and a-b). Note that no molecular clashes occur upon binding of all three Fabs. The upper panel shows the complex as viewed down the gp41 three-fold axis and the lower panel shows the complex in a side view indicating the orientations of the membrane anchors. The arrows show the directions of the viral membrane and of the fusion peptide that will be inserted into the target cell membrane.
Figure 4
Figure 4. HK20 occupies a hydrophobic pocket on HR1, which is filled by HR2 in the six helix bundle post fusion conformation.
(A) Close-up of the six helix bundle conformation of the HK20 epitope. The HR1 trimer is shown as a molecular surface (yellow) and the HR2 helix pointing residues Trp626, Trp631 and Ile635 into the pocket is shown as coil motif. (B) Close up of the HK20 CDR H2 pointing into the hydrophobic pocket together with the main structural principles of CDR H3 and L3 interactions. (C) Close-up of the D5 interaction sites reveals similar structural principles as shown for HK20 (see B).
Figure 5
Figure 5. Surface representation of the HK20 and D5 footprints on 5-Helix.
Interface area of HK20 (A) and of D5 (B). The HR1 surface is shown in yellow, the HR2 surface in grey and residues making direct contacts with HR1 are marked in red and HR2 in pink. Both HK20 and D5 interact indirectly with HR2 His643 via water-mediated hydrogen bonds.
Figure 6
Figure 6. Cα super positioning of the HK20-5-Helix and the D5-5-Helix complexes reveals differences in epitope approach angles.
(A) Cα super positioning of the 5-Helix HR1 helices A and C reveals differences in the approach angles of both complexes. While D5 binds in an orthogonal way to its HR1 epitope, HK20 approaches the epitope in an ∼60° angle. (B) The difference in binding angle is also supported by the Cα super positioning of VH and HR1 chains a and c. (C) Close-up of the super positioning of HK20 and D5 CDR H3 loops demonstrating their differences in interaction with the HR1 helix. (D) Close-up of the super positioning of HK20 and D5 CDR H2 loops highlights the central role of Phe54 contacting two HR1 helices.
Figure 7
Figure 7. Detection of HK20-like antibodies in patient sera.
(A) ELISA plates were coated with 5-Helix and incubated with serial plasma dilutions, followed by biotinylated HK20 mAb and by enzyme-conjugated streptavidin. Shown is the reciprocal plasma dilution that blocks 80% binding (BD80) of HK20 mAb. BD80 values <20 (empty circles) were scored as negative. Each symbol represents a different individual. (B) Total serum IgG binding to 5-Helix was determined by ELISA. Reciprocal EC50 values are shown. Empty circles correspond to plasma samples with BD80 values <20.
Figure 8
Figure 8. Comparison of HK20 IgG and D5 IgG in TZM-bl and HOS-based neutralization assays.
HK20 (circles) and D5 (squares) were tested for their capacity to neutralize the same 18 HIV-1 pseudoviruses representing 6 different clades in TZM-bl and HOS-cell based assays. Shown are nM IC50 values, (empty circles or squares, IC50 >2000 nM (>300 µg/ml).
Figure 9
Figure 9. Size dependent neutralization breadth and potency of HK20 in different HIV-1 neutralization assays and comparison with T-20.
(A) Neutralization of 45 HIV-1 isolates in TZM-bl cells by HK20 IgG (empty circles), Fab (red circles) and scFv (black circles). Shown are nM IC50 values. (B) Neutralization of 27 clade B and 25 clade C HIV-1 isolates in TZM-bl cells by HK20 scFv. Shown are nM IC50 values. Two-tailed P value was calculated with the unpaired t test. (C) HK20 scFv (black circles) and T-20 (empty triangles) were tested for their capacity to neutralize 20 HIV-1 isolates using either TZM-bl (left panel) or HOS (right panel) as target cells. Shown are nM IC50 values. The dotted line indicates the lowest concentration tested. (D) HK20 scFv (black circles), TriMab (2F5, 2G12 and b12, empty squares) and T-20 (empty triangles) were tested in a PBMC-based neutralization assay against a panel of 9 HIV-1 primary isolates from 5 different clades. Shown are nM IC90 values. The dotted lines indicate the highest concentration tested. Virus isolates used and detailed results are shown in Tables S1, S2, S3, S4, S5 in Supporting Information S1.
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