Anti-gp41 antibodies cloned from HIV-infected patients with broadly neutralizing serologic activity

Abstract

Most HIV-infected individuals develop antibodies to the gp120 and gp41 components of the viral spike; however, only a fraction of these individuals mount a broadly neutralizing serum response against HIV. We have cloned anti-HIV antibodies from the memory B-cell compartment of six individuals with variable viral loads and high titers of broadly neutralizing antibodies. Here, we report on the features of the anti-gp41 response in these patients. Competition experiments with previously characterized antibodies targeting defined epitopes on the gp41 ectodomain showed antibodies directed against the "immunodominant region" (cluster I), the carboxy-terminal heptad repeat (cluster II), and the membrane-proximal external region (cluster IV). On the other hand, antibodies directed against the amino-terminal part of the molecule, including the fusion peptide, polar region, and the N-terminal heptad repeat, were not detected. When all patients' data were combined, unique B-cell clones targeting cluster I, II, and IV accounted for 32%, 49%, and 53% of all anti-gp41-reactive B cells, respectively; therefore, no single region was truly immunodominant. Finally, although we found no new neutralizing epitopes or HIV-1-neutralizing activity by any of the gp41 antibodies at concentrations of up to 50 microg/ml, high concentrations of 7 out of 15 anti-cluster I antibodies neutralized tier 2 viruses.

FIG. 1.

HIV-1 gp41. (a) Diagrammatic representation of the gp41 ectodomain with the fusion peptide (FP), the polar region (PR), the amino-terminal and carboxy-terminal heptad repeat regions (NHR and CHR, respectively), the immunodominant region (ID), and the MPER (12). (b) The clusters I to VI are indicated. The amino acid residues (LAI sequence) are shown below the different clusters. Antibodies used in competition ELISAs are shown in italics.

FIG. 2.

gp41 ELISA. (a) The result of the IgG gp41 adsorption. The affinity-purified gp41 binding IgG fractions (pos) and the anti-gp41-depleted (neg) IgG fractions are shown for each of three different patients (Pt1 to Pt3), according to the legend on the figure. HC, healthy control; OD, optical density. (b) Graphs show lack of competition between the affinity-purified anti-gp41 serum IgG fractions and biotinylated 2F5 or 4E10. Controls are unbiotinylated 2F5 and 4E10 as well as HC IgG and HIV IG. (c) Competition between affinity-purified anti-gp41 serum IgG and anti-cluster I antibodies (2-55 and 2-378). (d) Results of the ELISAs with the 2F5 and 4E10 MPER-specific peptides. Fractions are colored as indicated on the figure.

FIG. 3.

Neutralizing activity. Shown is the increase or decrease in the neutralizing activity of IgG against different pseudoviruses for patients 1 to 3 after depletion of the gp41 binding antibodies by gp41 coupled to magnetic beads.

FIG. 4.

Clonal expansion and cluster distribution of anti-gp41 antibodies. Pie charts show the distribution of anti-gp41 antibodies to the different clusters (I, II, IV, and VI). Patients (Pt1 to Pt4) are indicated. The number in the center indicates the number of antibodies; slices are proportional to the size of the unique clones (43). The expanded cluster I antibodies are highlighted in red.

FIG. 5.

gp41 hot-spot inhibition map with IC₅₀s (μg/ml). ID, the competing antibody reacts with the immunodominant peptide; ND, not done. Representative ELISA inhibition graphs are also shown (b). Black lines indicate inhibition. OD, optical density; abs, antibodies.

FIG. 6.

Rare glyco-dependent binding by anti-gp41 antibodies. (a) Coomassie blue-stained polyacrylamide gel and Western blot analysis with MAb 2F5 of control and deglycosylated gp41. As specified by the manufacturer (Acris), the protein shows three major immunospecific bands between 20 and 30 kDa, minor bands between 20 and 30 kDa, bands at 14 and 7 kDa, and an aggregation smear at 35 kDa and above. The aggregation smear condenses in major specific bands after deglycosylation. (b) Deglycosylation was further confirmed by Lens culinaris lectin precipitation, followed by elution with 0.5 M methyl α-d-mannopyranoside of glycosylated gp41 but not of the deglycosylated form. (c) Graph summarizes differences in binding of anti-gp41 antibodies to intact (red bars) and deglycosylated (green bars) gp41 as measured by ELISA (OD at 405 nm). A significant decrease in binding is indicated by a black arrow.