Since HR-1 occupies a restricted surface only transiently exposed during the HIV-1 entry process, we hypothesized that accessibility of the HK20 target epitope might be limited by the size of an IgG molecule. We therefore compared intact IgG and Fab fragments of HK20 for their capacity to neutralize a panel of 22 pseudoviruses in the HOS-based assay. The HK20 Fab fragments showed markedly increased breadth and potency compared to IgG, being able to neutralize 21/22 isolates with IC 50 values ranging from 0.01 to 5.7 mg/ml ( Figure 5D -E and Table S4 ). By contrast, HK20 IgG neutralized 19/22 isolates with IC 50 values ranging from 1.46 to 84 mg/ml. In addition, when the HK20 Fab fragment was tested in the TZM-bl assay it neutralized 10 out of 33 isolates with IC 50 values ranging between 0.4 and 8.8 mg/ml (Table S2) , thus suggesting that the lack of activity observed with HK20 IgG was possibly associated to a different susceptibility of this cell line to this type of entry inhibitors. Overall, these findings suggest that the reduced size of the Fab molecule allows increased access to the HR-1 region and imply that HR-1 accessibility is largely dependent on the target cell used. This is similar in concept to the enhanced neutralization profiles of Fab and ScFv specific for the CD4i surface [39] . Further studies using M7 cells [52] challenged with replication-competent HIV-1 viruses carrying a Luc reporter showed that HK20 exhibits broad neutralization consistent with the results from the HOS cell assay but unlike the TZM-bl assay, being able to neutralize 4 out 5 viruses tested (Table S5) .