The three new neutralizing mAbs HJ16, HGN194 and HK20 show some interesting features. HJ16 showed a breadth of neutralizing activity comparable to, and generally complementary to b12, which aside from PG9 and PG16 [40] is currently the most potent of the relatively broad neutralizing antibodies available. HJ16 also showed selective neutralization of multiple Tier-2 isolates, making it particularly relevant as a template for vaccine design. HGN194 binds to an extremely conserved epitope in the V3 crown, and neutralizes all Tier-1, but only 11% of Tier-2 isolates tested. HGN194 showed broader activity than the well-characterized V3-specific mAb 447-52D against a panel of 92 pseudoviruses. The preference for Tier-1 isolates characteristic of V3-specific mAbs is consistent with the idea that the V3 loop is displayed to varying degrees in the context of the native trimeric Env protein of individual isolates [67, 68] and with the recent observation that sCD4 broadens neutralization of V3 mAbs [69] . The results obtained with HK20 are particularly intriguing, since this mAb has a broad pattern of neutralization observed with HOS but not TZMbl target cells. This mAb recognizes a highly conserved epitope in the HR-1 trimer and it is more effective as an Fab fragment as compared to intact IgG, a fact that is may be explained by the limited accessibility of the epitope, which is likely to be exposed only transiently on the cell surface during the viral entry process [70] . Intriguingly, a recent study indicates that in TZM-bl cells HIV-1 pseudoviruses penetrate the cytoplasm from within endosomes rather than from the cell surface [71] , a finding that might help explain the lack of activity of HK20 in the TZMbl assay if binding of this mAb is affected by the endosomal environment. Further studies on primary cells will be required to address the relative importance of these two entry pathways and to establish whether HK20 may be capable of exerting a broad and potent neutralizing activity in vivo. Preliminary studies using M7 cells [52] challenged with replication-competent HIV-1 viruses Cross competition with biotinylated mAbs of known specificity: +, 90-100% inhibition, +/2, 50-90% inhibition, 2, ,50% inhibition. Binding to different gp41 constructs by ELISA. Minimal linear epitopes using the Pepscan analysis and specificity assignment. C-C, C-C loop; 5HB, 5-helix bundle; FP, fusion peptideShown is also the number of isolates neutralized in the HOS-based and TZM-bl based assays. doi:10.1371/journal.pone.0008805.t002