Odies that could not be mapped to gp120 were responsible for neutralizing different viruses against which the plasma was tested. Two recent studies demonstrated that, indeed, the overall crossneutralizing activity in a chronically HIV-infected subject could be recapitulated by two monoclonal antibodies isolated from that subject, that target distinct epitopes on Env [42,43]. However, those studies did not examine whether both specificities emerged at the same time or not, and how they evolved over time. In the present study we aimed to better understand the relative emergence and evolution of dual epitope specificities in a wellcharacterized case control from the above-described cohort.?MAbs at a concentration of 10 mg/ml in naive plasma were performed as a positive control. 250 ml or 500 ml of plasma diluted 1:5 in DMEM/10 FBS were incubated with 100 ml or 200 ml Env protein-coupled beads, respectively, at 37uC for 120 min with gentle rotation. The samples were placed on a magnet and the beads were isolated. Four rounds of bead adsorptions were performed per sample. The anti-gp120 plasma antibodies bound to the bead-coupled Env proteins were eluted in a series of increasingly acidic solutions as previously described [24]. The beads from each serial adsorption were combined and incubated in 0.1 M GlycineHCl, pH 2.7 for 30 seconds with vortexing. The beads were collected by brief centrifugation and held in place by a magnet. The supernatant was removed and adjusted to pH 7.5 with 1 M Tris (pH 9.0). The process was repeated with the beads in 0.1 M Glycine-HCl, pH 2.3, and then again in pH 1.7. The final supernatants were buffer exchanged in PBS and washed over a 30 kD Amicon Ultra centrifugation concentrator (Millipore). Concentration of immunoglobulin was determined by absorbance at 280 nm (NanoDrop Spectrophotometer ND-1000, Thermo). The anti-gp120-antibody depleted plasmas and the anti-gp120 antibodies eluted from gp120-coated beads were tested by ELISA for binding activity, and for neutralizing activity (data not shown).Neutralization AssaysSingle round-competent viruses expressing Env’s from Clades A, B, and C primary isolates were used in this study. The clade B SF162, JRFL and YU2 isolates were isolated during chronic HIV1 infection, and the remaining isolates 24272870 were isolated during acute Lecirelin infection [44?6]. Single-round competent viruses were produced in 293 cells as previously described [47], with the modification that GeneJuice (EMD Millipore) was used as the transfection reagent. In the cases when kifunenisne- or swainsonine-treated pseudoviruses 1407003 were used, the 293 producer cells were treated with 50 mM of the glycosidase inhibitor after transfection and the pseudovirus was collected after 72 hour incubation. All treated pseudoviruses were tested for neutralization resistance to PG9 or PG16. The neutralizing activities of plasmas were determined using the Tzm-bl-based neutralization assay [48]. Briefly, plasma dilutions were pre-incubated with single-round competent virions (pseudovirus) for 90 minutes at 37uC. The plasma/pseudovirus mixture was added to TZM-bl cells (plated the day before at 3000 cells per well in a 96-well plate) for 72 hrs at 37uC. The supernatant was removed and 100 ml of Steady-Glo Luciferase Assay Substrate (Triptorelin web Promega) was added to each well. Plates were incubated for 15 minutes at room temperature away from light, and 75 ml of the lysate was transferred to micro titer plates. The cell-associated luciferase act.Odies that could not be mapped to gp120 were responsible for neutralizing different viruses against which the plasma was tested. Two recent studies demonstrated that, indeed, the overall crossneutralizing activity in a chronically HIV-infected subject could be recapitulated by two monoclonal antibodies isolated from that subject, that target distinct epitopes on Env [42,43]. However, those studies did not examine whether both specificities emerged at the same time or not, and how they evolved over time. In the present study we aimed to better understand the relative emergence and evolution of dual epitope specificities in a wellcharacterized case control from the above-described cohort.?MAbs at a concentration of 10 mg/ml in naive plasma were performed as a positive control. 250 ml or 500 ml of plasma diluted 1:5 in DMEM/10 FBS were incubated with 100 ml or 200 ml Env protein-coupled beads, respectively, at 37uC for 120 min with gentle rotation. The samples were placed on a magnet and the beads were isolated. Four rounds of bead adsorptions were performed per sample. The anti-gp120 plasma antibodies bound to the bead-coupled Env proteins were eluted in a series of increasingly acidic solutions as previously described [24]. The beads from each serial adsorption were combined and incubated in 0.1 M GlycineHCl, pH 2.7 for 30 seconds with vortexing. The beads were collected by brief centrifugation and held in place by a magnet. The supernatant was removed and adjusted to pH 7.5 with 1 M Tris (pH 9.0). The process was repeated with the beads in 0.1 M Glycine-HCl, pH 2.3, and then again in pH 1.7. The final supernatants were buffer exchanged in PBS and washed over a 30 kD Amicon Ultra centrifugation concentrator (Millipore). Concentration of immunoglobulin was determined by absorbance at 280 nm (NanoDrop Spectrophotometer ND-1000, Thermo). The anti-gp120-antibody depleted plasmas and the anti-gp120 antibodies eluted from gp120-coated beads were tested by ELISA for binding activity, and for neutralizing activity (data not shown).Neutralization AssaysSingle round-competent viruses expressing Env’s from Clades A, B, and C primary isolates were used in this study. The clade B SF162, JRFL and YU2 isolates were isolated during chronic HIV1 infection, and the remaining isolates 24272870 were isolated during acute infection [44?6]. Single-round competent viruses were produced in 293 cells as previously described [47], with the modification that GeneJuice (EMD Millipore) was used as the transfection reagent. In the cases when kifunenisne- or swainsonine-treated pseudoviruses 1407003 were used, the 293 producer cells were treated with 50 mM of the glycosidase inhibitor after transfection and the pseudovirus was collected after 72 hour incubation. All treated pseudoviruses were tested for neutralization resistance to PG9 or PG16. The neutralizing activities of plasmas were determined using the Tzm-bl-based neutralization assay [48]. Briefly, plasma dilutions were pre-incubated with single-round competent virions (pseudovirus) for 90 minutes at 37uC. The plasma/pseudovirus mixture was added to TZM-bl cells (plated the day before at 3000 cells per well in a 96-well plate) for 72 hrs at 37uC. The supernatant was removed and 100 ml of Steady-Glo Luciferase Assay Substrate (Promega) was added to each well. Plates were incubated for 15 minutes at room temperature away from light, and 75 ml of the lysate was transferred to micro titer plates. The cell-associated luciferase act.