Pseudovirus based assays. Neutralization capacity of patient plasma and mAbs against PV on TZMbl and the HOS cell related GHOST.CD4-X4/R5 cells was determined as described [13, 14] . Luciferase reporter gene activity was quantified 48-72 h after infection upon cell lysis and addition of firefly luciferase substrate (Perkin-Elmer) as described. Emitted relative light units (RLUs) were quantified on a LB941 Berthold luminometer (Alabama, US). Infection of TZMbl cells was quantified using SteadyLite and infection of GHOST cells was quantified using BriteLite as a substrate (both Perkin-Elmer). In a preliminary experiment 1.10 4 TZMbl or GHOST cells were seeded in each well of 96-well, flat bottom plates and infected with a range of viral doses in a total volume of 200 ml to establish the dose, which resulted in a signal of 50,000 to 100,000 RLU in the presence of 10 mg/ml diethylaminoethyl-dextran (DEAE-dextran, Sigma, Belgium) to enhance virus infectivity in TZMbl cells, while no DEAE-Dextran was used in GHOST cells. In the actual neutralization experiments, mAbs or plasma were pre-incubated with PV for 1 h at 37uC. The mAb concentration or plasma dilution producing a 50% reduction in luciferase reporter gene production was determined by linear regression analysis in Microsoft Office Excel as described on http://www.hiv.lanl.gov/content/nab-reference-strains/ html/Protocol-for-Neutralizing-Antibody-Screening-Assay-for-HIV-1in-TZM-bl-Cells-November-2010.pdf. For IC50 of mAbs, the 50% inhibitory concentrations were determined via a linear interpolation method using the mean of duplicate or triplicate cultures. The assay readouts for the dilutions above and below the IC50 were joined with a straight line, plotted against the log concentration of mAb. The position where the line crossed the 50% assay readout was taken as the IC50 estimate. Where the IC value was outside the range of concentrations tested, it was recorded as either greater than the highest concentration used, or less than the lowest concentration, as appropriate. An ID50 for plasma and IC50 for Abs were calculated from a dilution series starting from 1:20 for plasma and starting from 50 or 150 mg for Abs depending on the Ab used.