Role of HIV membrane in neutralization by two broadly neutralizing antibodies

Abstract

Induction of effective antibody responses against HIV-1 infection remains an elusive goal for vaccine development. Progress may require in-depth understanding of the molecular mechanisms of neutralization by monoclonal antibodies. We have analyzed the molecular actions of two rare, broadly neutralizing, human monoclonal antibodies, 4E10 and 2F5, which target the transiently exposed epitopes in the membrane proximal external region (MPER) of HIV-1 gp41 envelope during viral entry. Both have long CDR H3 loops with a hydrophobic surface facing away from the peptide epitope. We find that the hydrophobic residues of 4E10 mediate a reversible attachment to the viral membrane and that they are essential for neutralization, but not for interaction with gp41. We propose that these antibodies associate with the viral membrane in a required first step and are thereby poised to capture the transient gp41 fusion intermediate. These results bear directly on strategies for rational design of HIV-1 envelope immunogens.

Fig. 1.

Binding to synthetic lipid bilayer and HIV-1 membrane of the scFv from broadly neutralizing antibody 4E10. Liposomes that mimic the lipid composition of HIV viral membrane containing phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine/sphingomyelin/cholesterol with a ratio of 9.35:19.25:8.25:18.1:45 (24) (A) or AT-2 inactivated virion preparation of HIV-1 ADA (B) were immobilized at similar levels on L1 chips through an alkyl linker. 4E10 scFv and its variants were passed over the surface at a concentration of 8 μM (200 μg/mL). An anti-gp120 mAb, A32, and an anti-gp41 13H11, which show no binding to lipids (4), were also passed over the surface as negative controls. The sensorgrams of 4E10 in B were subtracted from the one obtained with A32. The recorded sensorgrams are shown in black for 4E10 scFv; dark red for 4E10-mut1 scFv; orange for 4E10-mut2 scFv; red for 4E10-mut3 scFv; blue for 4E10-mut4 scFv; light gray for mAb A32; and dark gray for 13H11 Fab. The experiments were repeated at least twice with similar results. Dissociation constants derived from the titration series using either PS-liposomes or HIV-1 virions, shown in Fig. S6, are summarized in (C). The weak affinities of 4E10-mut2 and 4E10-mut3 scFv could not be determined accurately. N.D., not determined.

Fig. 2.

Interaction of 4E10 scFv and its mutants with the HIV-1 gp41 prehairpin intermediate. 92UG-gp41-inter-Fd was immobilized on a CM5 chip. Each of the following analytes was passed over a freshly prepared, gp41-inter surface, as the chip could not be completely regenerated (see Materials and Methods): (A) 4E10 scFv (10 and 100 nM); (B) 4E10-mut1 scFv (10 and 100 nM); (C) 4E10-mut2 scFv (10 and 100 nM); and (D) 4E10-mut3 scFv (10 and 100 nM). (E) 4E10-mut4 scFv at various concentrations (10, 25, 50, 75, and 100 nM) was passed over a single chip regenerated between runs by a solution containing 50 mM NaOH and 2 M NaCl. Binding kinetics were evaluated using BIAevaluation software (Biacore) and a 1:1 Langmuir binding model. The recorded sensorgrams are shown in black and the fits in green. The derived constants are summarized in (F). All experiments were repeated at least three times using different preparations of the proteins and gave essentially the same results.

Fig. 3.

Neutralization by 4E10 variants and blocking of neutralization by gp41-inter. (A) Neutralization determined by reduction of luciferase reporter-gene expression after a single round of infection by pseudotyped HIV-1 viruses in TZM-bl cells. The pseudotyped viruses containing Envs derived from HIV-1 isolates, BG1168 and SF162, were preincubated with serial dilutions of the antibodies in 96-well plates. After mixing and incubation with target TZM-bl cells, the concentration of half-maximal inhibition (IC₅₀) was calculated from the luciferase activities determined by luminescence measurements. (B) Effect of addition of gp41-inter on neutralization by 4E10. Serial dilutions of 4E10 IgG were plated and preincubated with the BG1168 pseudovirus for 1 h at 37 °C, followed by addition of TZM-bl cells, premixed with either HIV-1 or SIV gp41-inter protein. Percent neutralization was determined from luciferase activities and plotted against the concentration of 4E10. Black, neutralization by 4E10 (no gp41-inter added); light blue and blue, neutralization by 4E10 in the presence of SIV gp41-inter (6.25 and 12.5 μg/mL, respectively); light red and red, neutralization in the presence of HIV gp41-inter (6.25 and 12.5 μg/mL, respectively). IC₅₀ values were read directly from the plots. The experiments were repeated twice and gave the same results.

Fig. 4.

Two-step model for neutralization of HIV-1 by MPER-directed antibodies. HIV envelope glycoprotein undergoes a large structural transition (from the prefusion state to the prehairpin intermediate conformation) after sequential binding of receptor, CD4, and coreceptor, CXCR4 or CCR5, to gp120. Dissociation of gp120 and insertion of the fusion peptide of gp41 into the target cell membrane lead to formation of a prehairpin intermediate. Antibody is shown in pink; gp120, in green; and the untriggered gp41 in blue. After conformational changes, the N-terminal fusion peptide of gp41 is shown in green; heptad repeat 1 (HR1) in yellow; the C-C loop in purple; heptad repeat 2 (HR2) in dark blue; MPER in red and the transmembrane segment in cyan. Step1: MPER-directed antibody (e.g., 4E10 or 2F5) attaches to the viral membrane through its long, hydrophobic CDR H3 loop. The MPER is either concealed or configured inappropriately for antibody recognition until the virus encounters CD4 and coreceptor. Step 2: Once triggered by association of gp120 with CD4 and coreceptor, gp41 undergoes a cascade of conformational changes, leading to the prehairpin intermediate. Antibodies such as 4E10 or 2F5 have only a brief interval within which their MPER target is exposed. Once the antibody has docked onto the gp41 epitope, it can prevent the further structural rearrangements of gp41 that are required for membrane fusion.