Fine Definition of the Epitope on the gp41 Glycoprotein of Human Immunodeficiency Virus Type 1 for the Neutralizing Monoclonal Antibody 2F5

Abstract

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

Neutralization Escape Variants of Human Immunodeficiency Virus Type 1 Are Transmitted from Mother to Infant

“…The MAb biz, which recognizes a complex epitope in the CD4-binding domain of envelope surface unit gp120 (36), neutralized 3 of the 10 variants tested, with IC 50 s of 0.8, 9.6, and 41.7 g/ml ( Table 2). The 2F5 and 4E10, which target the DKW and WFXI motifs, respectively, in the envelope transmembrane unit gp41 (3,32,34,43,49), neutralized nine and seven variants, respectively, with various potencies. Variants with changes within the antibody epitope (e.g., BF535.A1 and BJ412.S3) were resistant to neutralization by that MAb.…”

Structure and Immunogenicity of a Peptide Vaccine, Including the Complete HIV-1 gp41 2F5 Epitope

“…Fine definition of the 2F5 epitope suggests the involvement in antibody recognition of a helical stretch that follows the core epitope residues (34,36,63). It has been argued that the MAb2F5's CDR-H3 loop establishes contact with residues located further downstream within a continuous helix (25,38).…”

“…The MPER sequence below highlights the five Trp residues in green and the core epitope residues recognized by 2F5 antibody underlined. The line on top spans the extended 2F5 epitope as defined by proteomic analyses (34). Blue asterisks denote residues implied in secondary binding by CDR-H3 loop (25) and the box an aromatic rich anchor to the membrane interface.…”

“…Based on mass spectrometry and proteolytic protection assays, this core epitope was later extended to span the 656 NEQELLELDK-WASLWN 671 sequence (34). Comparable full epitope lengths were subsequently suggested by competition ELISA (35) and structural analyses (14,36).…”

Thermodynamic Analysis of the Binding of 2F5 (Fab and Immunoglobulin G Forms) to Its gp41 Epitope Reveals a Strong Influence of the Immunoglobulin Fc Region on Affinity

“…The core epitope of 2F5 was mapped onto the 662 ELDKWAS 668 linear sequence of gp41, which is located at the membrane-proximal external region (MPER) of the protein. A functional epitope including those residues whose substitution modifies the affinity for the corresponding antibody was later extended to the sequence 656 NEQELLELDK-WASLWN 671 (7). The atomic interactions of the complex between the core epitope and antibody 2F5 have been described in detail (8 -10).…”