In order to generate these mAbs, patient plasma were selected with a neutralization assay with an extended incubation time, using activated PBMC and a panel of clinically isolated replication competent HIV-1 strains. This assay differs from the classical 'short' PBMC neutralization assay by extending the incubation phase of plasma with virus from 1 to 24 hours. The importance of this format was shown in a SHIV challenge trial in rhesus macaques, where recombinant HIV envelope immunizations induced protection [5, 6] . Comparing various neutralization assays, we showed that the PBMC based assay with an extended incubation phase was able to discriminate between protected and non-protected animals after vaccination. Since we are attempting to develop a vaccine effective against a range of subtypes and because the subtype A, subtype C and circulating recombinant form (CRF) 02_AG are responsible for at least 75% of the current new infections worldwide, we identified patients, whose plasma could cross-neutralize mainly viruses from these three subtypes in the extended incubation PBMC assay. From the blood of selected patients, memory B cells were isolated and immortalized using an Epstein Barr Virus (EBV) based procedure [7] . Supernatants of B cell clones were tested in ELISA with recombinant gp41, trimeric gp120 and gp140 proteins from several subtypes as solid phase. Clones with binding activity to any of these antigens were expanded and supernatants were tested using a HOS based PV neutralization assay. This effort ultimately resulted in the selection of the new mAbs HJ16, HGN194 and HK20, which showed considerable breadth of neutralizing activity against a panel of HIV-1 primary isolates spanning both tier 1 and tier 2 viruses of different subtypes [2] .