Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), i n combination with proteolytic. protection assays, has been used to identif y the functional epitope on human immunodeficiency virus envelope glycoprot ein gp41 for the broadly neutralizing and-gp41 human monoclonal antibody 2F 5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subun its (SOS gp140) was affinity bound to immobilized 2F5 under physiological; conditions. A combination of proteolytic enzymatic cleavages was then perfo rmed to remove unprotected residues. Residues of SOS gp140 protected by the ir binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitop e, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is signi ficantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent. assay. This new knowledge of the st ructure of the 2F5 epitope may facilitate the design of vaccine antigens in tended to induce antibodies with the breadth and potency of action of the 2 F5 monoclonal antibody.