FURTHER CHARACTERIZATION OF AN ANTIGENIC SITE OF HIV-1 GP120 RECOGNIZED BY VIRUS NEUTRALIZING HUMAN MONOCLONAL-ANTIBODIES

Objective: The aim of this study is to characterize antigenic sites on HIV-1 gp120 which may be important for the development of active and passive immunization strategies against HIV-1 infection. Design: Two H IV-1-seropositive individuals were selected from the Amsterdam cohort and Epstein-Barr virus (EBV)-transformed B cells were generated from t heir peripheral blood mononuclear cells, which produce HIV-1-specific human monoclonal antibodies (HuMAb). Methods: HuMAb were generated and selected based on their reactivities with native gp120. Reactivity wi th HIV-1 strains from phylogenetically different subfamilies was deter mined by immunostaining and virus neutralization assays. Specificity f or the CD4-binding site was tested by an inhibition enzyme-linked immu nosorbent assay and amino acids (aa) involved in the binding of the Hu MAb were identified with a set of gp120 molecules with single aa subst itutions. Results: Three HuMAb (GP13, GP44, GP68) were generated, all recognizing a conserved conformation dependent epitope within, or topo graphically near, the CD4-binding site of gp120. HuMAb GP13 and GP68 n eutralized a broad range of HIV-1 strains from phylogenetically differ ent subfamilies, whereas HuMAb GP44 exhibited a more restricted patter n of neutralizing activity. The patterns of gp120 aa involved in their binding were unique for each of these HuMAb. Conclusions: The pattern of reactivities of these three HIV-1-neutralizing HuMAb developed in these studies is similar to, but distinct from other human and rodent MAb that recognize this antigenic site of HIV-1 gp120.