Fig. 2. PRO 2000 competitively inhibits gp120 mAb binding to X4 and R5 viruses.

In Figure 2A–2C, PRO 2000 inhibited the binding of mAbs to the V3 loop and the CD4-binding site, but had little effect on the mAb binding to the N-terminus of gp120. The capacity of PRO 2000 to inhibit mAb binding to soluble gp120 was tested in a blocking sandwich ELISA. Soluble gp120_LAI or gp120_JRFL (1 µg/ml) was treated with the designated concentrations of PRO 2000 and captured onto ELISA plate with sheep anti-C terminus of gp120. The binding of mAbs to the V3 (2A), CD4-binding site (2B), and N-terminal C1 region (2C) of PRO 2000-treated gp120 was detected by alkaline phosphatase-conjugated secondary anti-human IgG antibody. MAb binding to gp120 in the absence of PRO 2000 was considered to be 100%; the OD₄₀₅ values ranged from 1.0 to 2.9, depending on the combinations of gp120 and mAbs tested. Background levels (0% binding) were determined from wells reacted with an irrelevant mAb control. Data from one of two experiments are shown. In Figure 2D and 2E, PRO 2000 inhibited the binding of mAbs to V3 and the CD4-binding site of gp120 expressed on HIV-1 virions. Virus capture assay was performed to assess the inhibitory effect of PRO 2000 on mAb binding to intact HIV pseudovirions. Pseudovirions bearing envelope of HXB2 (50 ng p24/ml) or JRFL (100 ng p24/ml) isolates were treated with varying concentrations of PRO 2000 and captured by V3 mAb 447-52D (2D), CD4-binding site mAb b12 (2E), or mAb to the C-terminal C5 region (2F) coated on ELISA plates. The amount of p24 associated with the captured virions was determined by non-commercial p24 ELISA. The quantity of virions captured in the absence of PRO 2000 was considered as 100% (typically 40–100 pg p24/ml for HXB2 and 100–200 pg p24/ml for JRFL). Background levels were obtained from wells containing no p24 or virions or from wells in which virions were captured with an irrelevant mAb. The data shown are from one of 3 or 4 repeated experiments.