Article Figures & Data
Figures
- Fig. 1.
MAb reactivity with epitopes in the gp120 coreceptor-binding domain during cell-cell fusion. CellTracker Green-labeled Env cells were cocultivated with target cells for the indicated times as described in Materials and Methods. Immunostaining was performed with BS3-fixed cells or with unfixed cells cooled to 4°C (noted at right) as described in Materials and Methods. The indicated MAbs were used at 5 μg/ml. Cell nuclei appear as gray areas in stained syncytia. Fusion of Env and target cells produces the light green cytoplasmic staining surrounding the nuclei. All antibodies were tested in parallel along with human isotype controls, which produced no binding signal (data not shown). Reactivity was detected only with the unfixed cells (right); due to the faint signal, the images were digitally enhanced for clarity. Arrows indicate areas of Env cell staining that are not in contact with target cells. Representative images are shown. Each experiment was repeated at least three times with the same results. Scale bar, 10 μm.
- Fig. 2.
MAbs 17b, 48d, and CG10 bind to HIV envelope-expressing cells in the presence of sCD4. Env cells were either untreated or incubated with 1 μg of sCD4/ml in DMEM for 120 min at 4°C and then either fixed with BS3 or directly stained with the indicated MAbs (visible in red) as described in Materials and Methods. MAbs were tested in parallel, along with human isotype controls, which produced no binding signal (data not shown). Representative images are shown. Each experiment was repeated at least three times with the same results. Scale bar, 10 μm.
- Fig. 3.
SDF-1 blocks cell-cell fusion but does not enable 17b binding. CellTracker Green-labeled Env cells and target cells were pretreated with 3 μg of SDF-1/ml for 1 h at 37°C prior to cocultivation. The cells were then incubated for the indicated times as described in Materials and Methods in the presence of 3 μg of SDF-1/ml. Fusing cells were either fixed with BS3 or cooled to 4°C and immunostained with 17b at 5 μg/ml. Reactivity was detected only with the unfixed cells; due to the faint signal, the images were digitally enhanced for clarity. Representative images are shown. Each experiment was repeated at least three times with the same results. Scale bar, 10 μm.
- Fig. 4.
17b Fab and MAbs directed against the gp120 coreceptor-binding site do not inhibit HIV-mediated cell-cell fusion. Neutralization activity was assayed by using Env and target cells as described in Materials and Methods. Env cells (104) were preincubated in triplicate wells with threefold serial dilutions of the indicated MAbs or Fab, starting at 100 μg/ml. The treated Env cells were then added to 104 target cells. Negative control assays were carried out in the absence of antibody or with normal human IgG (hIgG); positive control experiments included the broadly neutralizing MAb 2G12 (21). Cell-cell infection was determined after incubation at 37°C for 18 h by quantitative β-galactosidase assay. The percent inhibition of fusion for each test condition was calculated relative to control assays carried out in the absence of antibody. The averages of triplicate assays are shown.
- Fig. 5.
Temporal expression of the 8F101 and A32 epitopes on gp120 during cell-cell fusion. CellTracker Green-labeled Env cells were cocultivated with target cells for the indicated times as described in Materials and Methods. Immunostaining was performed with BS3-fixed cells as described in the text by using the MAbs 8F101 and A32 (visible in red) at 5 and 1 μg/ml, respectively. Two images are shown for each MAb at 30 min. One image depicts Env cells that have not yet undergone fusion, as indicated by the lack of cytoplasmic dye transfer. The second image depicts the MAb staining pattern observed after cell-cell fusion. Arrows indicates patches of 8F101 and A32 staining on unfused cells present 30 min after cocultivation. MAbs were tested in parallel, along with human and murine isotype controls. Isotype controls produced no binding signal (data not shown). Representative images are shown. Each experiment was repeated at least three separate times with the same results. Scale bar, 10 μm. In contrast to 17b, 48d, and CG10 (Fig. 1), the relatively strong binding signals obtained with these antibodies did not require digital enhancement.
- Fig. 6.
Effects of fusion inhibitors on 8F101 epitope exposure. (A) CellTracker Green-labeled Env cells and target cells were cocultivated for 120 min in the presence of 5 μg of T21/ml. Interacting cells were fixed with BS3 and immunostained with 8F101 at 5 μg/ml. Arrows indicate patches of MAb staining (visible in red) on the cell-cell interface. Representative images are shown. Each experiment was repeated at least three times with the same results. Scale bar, 10 μm. (B) CellTracker Green-labeled Env cells and target cells were pretreated with 3 μg of SDF-1/ml for 1 h at 37°C prior to cocultivation. The cells were then incubated for the indicated times as described in Materials and Methods in the presence of 3 μg of SDF-1/ml. Interacting cells were fixed with BS3 and immunostained with 8F101 at 5 μg/ml. Representative images are shown. Each experiment was repeated at least three times with the same results. Scale bar, 10 μm.
