Inhibition of HIV Env binding to cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged gp120

Abstract

During natural HIV infection, an array of host receptors are thought to influence virus attachment and the kinetics of infection. In this study, to probe the interactions of HIV envelope (Env) with various receptors, we assessed the inhibitory properties of various anti-Env monoclonal antibodies (mAbs) in binding assays. To assist in detecting Env in attachment assays, we generated Fc fusions of full-length wild-type gp120 and several variable loop-deleted gp120s. Through investigation of the inhibition of Env binding to cell lines expressing CD4, CCR5, DC-SIGN, syndecans or combinations thereof, we found that the broadly neutralizing mAb, 2G12, directed to a unique carbohydrate epitope of gp120, inhibited Env-CCR5 binding, partially inhibited Env-DC-SIGN binding, but had no effect on Env-syndecan association. Furthermore, 2G12 inhibited Env attachment to primary monocyte-derived dendritic cells, that expressed CD4 and CCR5 primary HIV receptors, as well as DC-SIGN, and suggested that the dual activities of 2G12 could be valuable in vivo for inhibiting initial virus dissemination and propagation.

Figure 1

Design and production of the Fc-gp120 chimeras. Fc-gp120 chimeras were constructed by fusing the C-terminus of human IgG1 Fc (H, CH2, CH3 domains) in-frame with the N-terminus of gp120_JR-CSF. Full-length wild-type (WT) and ΔV3, ΔV1V2 and ΔV1V2V3 forms of Fc-gp120 were expressed. A) SDS PAGE and Coomassie staining of purified Fc-gp120 chimeras under reducing and non-reducing conditions. B) ELISA assays comparing mAb binding to Fc-gp120 chimeras, an Fc control and monomeric full-length gp120 WT and ΔV1V2V3. Symbols are as indicated. Results are representative of two independent assays.

Figure 2

Binding of Fc-gp120 chimeras to cellular CD4. Inhibition of binding of Fc-gp120 chimeras to CEM-CD4 cells by (A) sCD4, (B) Fab b12 or (C) Fab 2G12. Inhibition of binding was assessed by FACS analysis and is expressed as % binding inhibition. Results are means of triplicate determinations.

Figure 3

Binding of Fc-gp120 chimeras to cellular CCR5. A) Inhibition of Fc-gp120-sCD4 complex binding to CCR5 by RANTES (1 μg/ml), TAK779 (0.5 μg/ml) and 2G12. Binding inhibition was assessed by FACS analysis and is expressed as a percentage calculated with reference to m.f.i. data using Fc-gp120-sCD4 complexes alone (0% inhibition value) and background with nothing added (100% inhibition value). Results are the means of triplicates. B) Inhibition assays were performed using Fc-gp120 complexed with 1 or 10 μg/ml of sCD4 and mAbs 2G12 or b12 at 10 or 25 μg/ml. Inhibition of binding was expressed as a percentage, as above. Results are the means of duplicates.

Figure 4

2G12 neutralizes HIV-1 JR-CSF effectively in a post-CD4 assay format. The neutralization activity of mAbs A) b12, B) X5, and C) 2G12 was assessed in the standard (closed circles) and post-CD4 (open circles) neutralization formats. Results are expressed as % of residual infection, with 100% representing infection in the absence of mAb. Results are representative of two experiments.

Figure 5

Effect of 2G12 on Env-HSPG and Env-DC-SIGN binding. A) The inhibitory effect of mannan (50 μg/ml) and 2G12 Fab (25 μg/ml) and 2G12 IgG (25 μg/ml) on binding of Fc-gp120 and gp120-Fc chimeras to CHO pgsA745 cells transduced with MIGR1 GFP/DC-SIGN. Fc alone was included as a negative control. Data are representative of triplicates. B) Namalwa cells expressing either CD4, HSPGs (syndecan 2) or DC-SIGN were assessed for Fc-gp120 binding in the absence or presence of varying concentrations of mAbs b12 and 2G12. Inhibition is expressed as a percentage of Fc-gp120 binding compared to controls. C) Virus capture by Namalwa cells expressing either HSPGs or DC-SIGN was assessed in the absence or presence of varying concentrations of mAbs b12 and 2G12. Inhibition is expressed as percentage of p24 captured compared to controls. Results are representative of two experiments.

Figure 6

2G12 inhibits DC-SIGN-gp120 interaction in ELISA. The ability of mAbs to inhibit DC-SIGN-gp120 interaction was evaluated. Fc-DC-SIGN was coated on the plate. Fixed concentrations of mAbs or mannan were then added, followed by graded concentrations of biotinylated Fc-gp120, which was detected using a streptavidin-alkaline phosphatase conjugate. Results are representative of two independent assays.

Figure 7

Inhibition of trans-infection by MDDCs and of Fc-gp120 binding to primary PBLs and MDDCs. A) MAbs b12 or 2G12, or mannan were incubated with virus (10 and 50 ng p24). The mixture was then incubated with primary MDCCs, followed by washing. Cells were then added on top of TZM-BL cells previously plated a day before, and trans-infection was assayed two days later by a β-gal assay. Results are the mean values of triplicates. B) Inhibition of Fc-gp120 binding to primary PBLs and MDDCs in the presence of sCD4, b12 Fab, 2G12 Fab or mannan at the concentrations indicated. Inhibition is expressed as the percentage of Fc-gp120 binding.