NEUTRALIZING RECOMBINANT HUMAN-ANTIBODIES TO A CONFORMATIONAL V2-BINDING AND CD4-BINDING SITE-SENSITIVE EPITOPE OF HIV-1 GP120 ISOLATED BY USING AN EPITOPE-MASKING PROCEDURE

As part of the goal of assembling a mixture of neutralizing human mAbs for possible prophylaxis and therapy of HIV-1 disease, we describe a strategy by which neutralizing human Abs to a weakly immunogenic epito pe can be accessed. From a phage display library derived from an asymp tomatic HIV-1 seropositive donor, a panel of recombinant Fabs against the CD4 binding site (CD4bs) of gp120 was retrieved by affinity select ion using recombinant gp120 (strain LAI). Two Fabs corresponding to th e dominant clones were used to mask the CD4bs epitope(s) before repeat ing the selection procedure. Four Fabs were then retrieved that had no vel heavy chain sequences. Three recognized a novel epitope distinct f rom that recognized by conventional CD4bs Abs and were defined by the following criteria: 1) second V region (V2 region) dependence indicate d by sensitivity to amino acid changes in the V2 loop and by competiti on with murine anti-V2 mAbs; 2) CD4bs dependence indicated by sensitiv ity to amino acid changes usually associated with CD4 binding and by i nhibition of Fab binding to gp120 by soluble CD4; this dependence seem ed to arise via conformational changes rather than by direct binding, as CD4bs Abs enhanced binding of two of the novel Fabs and, in a rever sal of the competition format, the novel Fabs did not inhibit soluble CD4 binding to gp120; and 3) equivalent binding to glycosylated and de glycosylated gp120 and significant, although much reduced, binding to denatured gp120 in contrast with CD4bs Abs, which do not bind to degly cosylated or denatured gp120. One of the novel Fabs efficiently neutra lized the MN and LAI strains of HIV-1. These results indicate the pres ence of a novel neutralizing conformational epitope on gp120 sensitive to the V2 loop and the CD4bs and further highlight the conformational flexibility of gp120. The strategy of masking highly immunogenic epit opes with Abs to rescue a broader range of specific Abs from combinato rial libraries should be widely applicable.