A fusion-intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies

Abstract

Most antibodies induced by HIV-1 are ineffective at preventing initiation or spread of infection because they are either nonneutralizing or narrowly isolate-specific. Rare, "broadly neutralizing" antibodies have been detected that recognize relatively conserved regions on the envelope glycoprotein. Using stringently characterized, homogeneous preparations of trimeric HIV-1 envelope protein in relevant conformations, we have analyzed the molecular mechanism of neutralization by two of these antibodies, 2F5 and 4E10. We find that their epitopes, in the membrane-proximal segment of the envelope protein ectodomain, are exposed only on a form designed to mimic an intermediate state during viral entry. These results help explain the rarity of 2F5- and 4E10-like antibody responses and suggest a strategy for eliciting them.

Fig. 1.

Expression constructs. (Upper) Schematic representations of HIV-1 envelope glycoproteins; gp160, the full-length precursor. Various 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, immunodominant loop with a conserved disulfide bond; HR2, heptad repeat 2; (see SI Fig. 9 for alignment); TM, transmembrane anchor; CT, cytoplasmic tail. Expression constructs are: gp140, uncleaved ectodomain of gp160 with a C-terminal His tag; gp140-Fd, uncleaved ectodomain of gp160 with a trimerization tag and a C-terminal His tag; gp41-inter, gp41 in the prehairpin intermediate conformation trapped by an N-terminal HR2 peptide and a C-terminal foldon tag; gp41-post, gp41 in the six-helix conformation with partial MPER. Glycans are represented by tree-like symbols. (Lower) Diagrams represent 3D organization of these protein species. Gp120 and gp41 in the prefusion state are shown in light green and light blue, respectively. The viral membrane is in orange. Other regions are colored as in the schematics above.

Fig. 2.

Preparations of the prefusion and the prehairpin intermediate conformations of HIV-1 gp41. (A) Purified HIV-1 92UG-gp140-Fd trimer was resolved by gel-filtration chromatography on Superose 6. The apparent molecular mass was calculated by using as standards thyroglobulin (670 kDa), ferritin (440 kDa), and catalase (232 kDa). (Inset) Peak fractions were pooled and analyzed by Coomassie blue-stained SDS/PAGE. (B) Sedimentation equilibrium of 92UG-gp140-Fd trimer with a Beckman XL-A analytical ultracentrifuge at 4°C. Three protein concentrations (0.62, 1.24, 2.48 μM) and three rotor speeds (1,820, 3,567, and 5,897 × g) were used. The data shown were collected with the protein at 1.24 μM and rotor speed of 3,567 × g. Data were analyzed with a single-species model; partial specific volume was calculated as 0.686 ml/g, using the sugar content. The molecular mass is 409 ± 10 kDa. (C) 92UG-gp140-Fd trimer was treated with various concentrations (lanes 1–7, 0, 0.05, 0.25, 0.5, 1, 2, 5 mM) of ethylene glycol bis(succinimidylsuccinate). Cross-linked products were analyzed by SDS/PAGE in a 4% gel. The molecular mass standard was cross-linked phosphorylase b (Sigma). Dimeric and trimeric species of 92UG-gp140-Fd migrate faster than expected for their molecular mass, probably because of compactness after cross-linking. (D) 92UG-gp41-inter was expressed in E. coli and refolded in vitro. (Inset) The refolded protein was resolved by gel-filtration chromatography on Superdex 200. It migrates on SDS/PAGE at molecular mass ≈26 kDa when sample is boiled and reduced; when not boiled and not reduced, there is a ladder of three bands (≈26, 50, 80 kDa, respectively), corresponding to monomer, dimer, and trimer. (E) Sedimentation equilibrium of 92UG-gp41-inter with a Beckman XL-A analytical ultracentrifuge at 4°C. Three protein concentrations (0.98, 1.96, 3.92 μM) and three rotor speeds (16,380, 22,295, and 49,213 × g) were used. Data shown were collected with the protein at 3.92 μM and rotor speed of 22,295 × g. Data were analyzed with a single-species model; the molecular mass is 92 ± 6 kDa. (F) 92UG-gp41-inter examined by negative-stain electron microscopy. Raw image of a field (Upper) and selected images after class averaging to increase signal-to-noise (Lower). The dimensions of the rod-like molecules are ≈150 Å × 45 Å. (Scale bar: 20 nm.)

Fig. 3.

mAb 2F5 binds the gp41 prehairpin intermediate. (A) 2F5 Fab was immobilized on a CM-5 chip, and 92UG-gp140-Fd (1 μM) or 92UG-gp140 (without foldon tag, 1 μM) was the analyte. The sensorgram for 92UG-gp140-Fd is shown in pink, 92UG-gp140-Fd at 37°C in orange, and 92UG-gp140 in red. Plasmin-cleaved 92UG-gp140-Fd was purified by gel-filtration chromatography on a preparation-grade Superdex 200 column; the fraction containing cleaved, trimeric gp140-Fd was immobilized on a Ni-NTA chip (see SI Methods); 2F5 Fab at 1 μM was the analyte; the sensorgram is shown in black. mAb 2F5 does not bind to any of the gp140 proteins. (Inset) 2F5 does react on an immunoblot with 92UG-gp140-Fd (lane 1), with the two gp41 proteins in the prehairpin intermediate conformation, 92UG-gp41-inter-Fd (lane 2), and HXB2-gp41-inter-GCN4 (lane 3). (B) The Fab fragment of 2F5 was immobilized on a CM-5 chip. Solutions at various concentrations of 92UG-gp41-inter-Fd, the gp41-inter protein derived from the 92UG037.8 sequence with a foldon tag, were the analyte. Binding kinetics were evaluated with a 1:1 Langmuir model using BiaEvaluation software (Biacore). The recorded sensorgrams are shown in black and the fits in green. (C) The Fab fragment of 2F5 was immobilized on a CM-5 chip. Solutions of 92UG-gp41-post at various concentrations (1.0, 2.5, 5.0, and 10.0 μM) were the analyte. The recorded sensorgrams are in black for 92UG-gp41-post and in green for the fits. (D) T20 peptide at different concentrations (5, 10, 25, and 50 nM) was the analyte with a chip bearing immobilized 2F5 Fab. The recorded sensorgrams are in black and the fits in green. Injections were carried out in duplicate and gave essentially the same results. Only one of the duplicates is shown.

Fig. 4.

mAb 4E10 binds the gp41 prehairpin intermediate. (A) 92UG-gp140-Fd trimer was immobilized on a Ni-NTA chip, and 4E10 IgG, 4E10 Fab, and 240-D IgG, all at 1 μM, were passed over the surface sequentially. Regeneration was not necessary after binding by 4E10 IgG and Fab. The recorded sensorgrams are in blue for 240-D, in pink for 4E10 IgG, and in red for 4E10 Fab. The 4E10 IgG binds only weakly, even with a potential avidity effect. (B) 92UG-gp41-inter was immobilized on a CM-5 chip. 4E10 scFv (50 nM) was the analyte. A duplicate run with the same chip gave lower binding because of the harsh regeneration conditions, but when repeated with a different chip, the results duplicated those shown here. The recorded sensorgrams are in black for 4E10 scFv and in green for the fit. (C) Western blot of 92UG-gp140-Fd trimer and 92UG-gp41-inter detected by mAb 4E10. Both 92UG-gp140-Fd (lane 1) and 92UG-gp41-inter (lane 2) react with 4E10. (D) Solutions of 4E10 scFv at various concentrations (25–500 nM) were passed over the surface of a SA chip bearing immobilized biotinylated 4E10 epitope peptide. The recorded sensorgrams are in black and the fits in green. All injections were carried out in duplicate and gave essentially the same results (except as described in B). Only one of the duplicates is shown in the figure.