Distinct conformational states of HIV-1 gp41 are recognized by neutralizing and non-neutralizing antibodies

Abstract

HIV-1 envelope glycoprotein gp41 undergoes large conformational changes to drive fusion of viral and target cell membranes, adopting at least three distinct conformations during the viral entry process. Neutralizing antibodies against gp41 block HIV-1 infection by targeting gp41's membrane-proximal external region in a fusion-intermediate state. Here we report biochemical and structural evidence that non-neutralizing antibodies, capable of binding with high affinity to an immunodominant segment adjacent to the neutralizing epitopes in the membrane-proximal region, recognize a gp41 conformation that exists only when membrane fusion is complete. We propose that these non-neutralizing antibodies are induced in HIV-1-infected individuals by gp41 in a triggered, postfusion form and contribute to production of ineffective humoral responses. These results have important implications for gp41-based vaccine design.

Figure 1. HIV-1 envelope constructs and GCN4-gp41-inter

Top, schematic representation of HIV-1 envelope glycoprotein gp160, the full-length precursor. Segments of gp120 and gp41 are designated as follows: C1–C5, conserved regions 1–5; V1–V5, variable regions 1–5; F, fusion peptide; HR1, heptad repeat 1; C-C loop, the immunodominant loop with a conserved disulfide bond; HR2, heptad repeat 2; MPER, membrane proximal external region; TM, transmembrane anchor; CT, cytoplasmic tail. Glycans are represented by tree-like symbols. HIV-1 envelope constructs used in this study include gp140, the uncleaved ectodomain of gp160 with a trimerization foldon (Fd) tag and a His-tag at its C-terminus; gp41-post, gp41 in the six helix conformation with partial MPER; gp41-inter, HR2 peptide- and foldon tag-trapped gp41 in the prehairpin intermediate conformation; GCN4-gp41-inter, gp41-inter with the six helix bundle portion replaced with a trimeric GCN4 coiled-coil (in light blue). Bottom, diagrams representing 3-D organization of gp41-inter and GCN4-gp41-inter. The trimeric GCN4 with its heptad repeat in the same register as HR1 replaces the HR2-linker-HR1 of gp41-inter. The coordinates of HR1 and GCN4 coiled-coils are shown in yellow and light blue, respectively.

Figure 2. Anti-HIV-1 gp41 cluster II antibodies preferentially bind gp41 in its postfusion conformation

Human anti-gp41 cluster II mAbs, 1281, 98-6D, 126-7D, 167D and 1379, were analyzed by a surface plasmon resonance (SPR) assay for binding to HIV-1 gp41 constructs: gp140 (sensorgrams in black); GNC4-gp41-inter (blue); and gp41-post (red). GCN4-gp41-inter or gp41-post was immobilized on CM5 chips; gp140 was captured on a Ni-NTA chip. Each IgG at 50 nM was passed over each surface individually. Data with the antibodies immobilized on a Protein A chip are shown in Supplementary Figure 3.

Figure 3. Analysis of interactions of 1281 Fab with various gp41 constructs

Fab fragment derived from mAb 1281 was tested by SPR for binding to gp41 constructs. (a) The recorded sensorgram for gp41-post is in red, gp140 in black and GCN4-gp41-inter in blue. (b) To confirm no detectable binding of 1281 Fab to GCN4-gp41-inter, solutions of 1281 Fab at various concentrations were flowed over the GCN4-gp41-inter surface. The sensorgrams are shown in various colors. In c and d, 1281 Fab at various concentrations were passed over the surfaces immobilized with gp41-post, and gp41-inter containing the six-helix bundle, respectively. Binding kinetics were evaluated using a 1:1 Langmuir binding model and binding constants are summarized in Supplementary Table 1. The sensorgrams are shown in black and the fits in green. All injections were carried out in duplicate and gave essentially identical results. Only one of the duplicates is shown.

Figure 4. Crystal structure of the complex of gp41-post and the Fab fragment of cluster II antibody 1281

Side (in a) and top (in b) views of the overall structure of the postfusion conformation of HIV-1 gp41 in complex with the Fab derived from an anti-gp41 cluster II mAb 1281 are shown in ribbon representation. The heavy chain of the antibody is in dark green and the light chain in light green; HR1 of gp41 in yellow, HR2 in blue and the part of MPER in red. The Fab mainly grips HR2, but also makes direct contacts with HR1 by CDR loops from both the heavy- and light- chains, suggesting the six-helix bundle conformation of gp41 is critical for 1281 binding. The MPER part in red contains the 2F5 epitope (residues 663–669), which is α-helical in the postfusion conformation.

Figure 5. Close-up of major contacts between gp41 and 1281 Fab

Gp41 and1281 Fab are both shown in ribbon diagram in a; gp41 in surface representation and the Fab in ribbon diagram in b. The heavy chain of the antibody is in dark green and the light chain in light green; HR1 of gp41 in yellow, HR2 in blue and the part of MPER in red; surface-exposed residues in HR2 are labeled in white. The CDR H1 and L2 loops of the antibody contact the HR2 helix in gp41-post; the CDR H3 reaches out and interacts with both the HR1 and HR2 helices. The footprint of the antibody covers residues 643–661, consistent with the previous epitope-mapping data,,. The 2F5 in red is spatially close to the cluster II epitope.