Patient plasma samples were evaluated for neutralizing antibody activity against virions pseudotyped with autologous patientderived viral Envs using a single round reporter assay described previously [17, 45] . Briefly, JC53BL-13 (Tzm-bl) cells were plated and cultured overnight. Two thousand infectious units of each pseudovirus was combined with five-fold dilutions of heatinactivated patient plasma and incubated for 1 hour at 37uC. Normal heat-inactivated human plasma was added as necessary to maintain a constant overall concentration. The virus-Ab mixture was then added to JC53BL-13 cells, and after two days, the cells were lysed, and the luciferase activity of each well was measured using a luminometer. Background luminescence was determined in uninfected wells and subtracted from all experimental wells. Percent infectivity was calculated by dividing the number of luciferase units at each plasma dilution by the value in the well containing no test plasma. The dilution of patient plasma that inhibited 50% of virus infectivity (IC50 titer) was determined using the Microsoft Excel 2004 for Mac Growth Function. Each experiment was performed independently at least twice with duplicate wells.