The correct and reliable determination of type I IFN biological activity is an important issue. The new bioassay presented in this study represents an attractive alternative to conventional type I IFN bioassays that work with cytotoxic live viruses. VSV*DG(Luc) is easily produced and handled under BSL-1 conditions. The activity of the replicon can be easily determined and standardized taking advantage of the two reporter proteins. It may be stored for prolonged time in lyophilized form without losing its activity. Finally, the proven sensitivity, rapidity, and accurateness of the assay recommends it for the determination of type I IFN from a number of mammalian and avian species. Finally, the test may be further developed to quantify the antiviral activity of other cytokines such as type II and type III IFNs. Characterization of Neutralizing Profiles in HIV-1 Infected Patients from whom the HJ16, HGN194 and HK20 mAbs were Obtained Several new human monoclonal antibodies (mAbs) with a neutralizing potential across different subtypes have recently been described. Three mAbs, HJ16, HGN194 and HK20, were obtained from patients within the HIV-1 cohort of the Institute of Tropical Medicine (ITM). Our aim was to generate immunization antibodies equivalent to those seen in plasma. Here, we describe the selection and characterization of patient plasma and their mAbs, using a range of neutralization assays, including several peripheral blood mononuclear cell (PBMC) based assays and replicating primary viruses as well as cell line based assays and pseudoviruses (PV). The principal criterion for selection of patient plasma was the activity in an ‘extended incubation phase’ PBMC assay. Neutralizing Abs, derived from their memory B cells, were then selected by ELISA with envelope proteins as solid phase. MAbs were subsequently tested in a high-throughput HOS-PV assay to assess functional neutralization. The present study indicates that the strong profiles in the patients' plasma were not solely due to antibodies represented by the newly isolated mAbs. Although results from the various assays were divergent, they by and large indicate that neutralizing Abs to other epitopes of the HIV-1 envelope are present in the plasma and synergy between Abs may be important. Thus, the spectrum of the obtained mAbs does not cover the range of cross-reactivity seen in plasma in these carefully selected patients irrespective of which neutralization assay is used. Nevertheless, these mAbs are relevant for immunogen discovery because they bind to the recombinant glycoproteins to which the immune response needs to be targeted in vivo. Our observations illustrate the remaining challenges required for successful immunogen design and development. Despite intense research efforts over nearly three decades, only minimal progress has been made in developing an HIV-1 vaccine. In retrospect, a number of reasons can be proposed for this failure such as the enormous genetic diversity of HIV, the camouflage of the neutralizing epitopes in the envelope spike by glycan shields, the presence of ''decoy'' immunodominant non-neutralizing antigenic determinants in non-conserved areas on the surface and the low gp120 trimer spike density on the virus membrane [1] . In addition, the most vulnerable regions may only be accessible for a short period. These short-lived structures include the so-called CD4 induced (CD4i) in gp120 and the pre-hairpin epitopes in gp41 that are only exposed following CD4 receptor binding and the subsequent conformational changes. Still, a few antibodies (Abs) are able to successfully interfere with the binding and fusion process, as seen in passive immunization studies in the macaque model. Such mAbs include 2G12 (binds to mannose residues on gp120); b12 and F105 (bind to the CD4 binding site, CD4bs); 17b and 65 (recognize conformational epitopes in the CD4i region); and 4E10 and 2F5 (bind to epitopes in the membrane proximal extracellular region or MPER of gp41). Last year, however, three new mAbs (HJ16, HGN194 and HK20) were reported from African patients from the ITM HIV-1 cohort. Taken together these mAbs target three different steps in viral entry: binding to CD4bs and thus preventing interaction of HIV-1 with CD4 by HJ16, binding to V3 and blocking the coreceptor binding by HGN194 and finally immobilizing the unfolding of the gp41 by the HK20 mAb [2] . Since HK20 targets HR1 instead of MPER or glycans in this region, it has the conceptual advantage over 4E10 and 2F5 of avoiding potential auto reactivity [2, 3] . Importantly, the HGN194 mAb has recently been found to confer protection in infant rhesus monkeys by the group of Ruprecht [4] .