Structure−affinity relationships in the gp41 ELDKWA epitope for the HIV‐1 neutralizing monoclonal antibody 2F5: effects of side‐chain and backbone modifications and conformational constraints

Abstract

The human monoclonal antibody, mAb 2F5, has broad HIV-1 neutralizing activity and binds a conserved linear epitope within the envelope glycoprotein gp41 having a core recognition sequence ELDKWA. In this study, the structural requirements of this epitope for high-affinity binding to mAb 2F5 were explored using peptide synthesis and competitive enzyme-linked immunosorbant assay (ELISA). Expansion of the minimal epitope to an end-capped, linear nonapeptide, Ac-LELDKWASL-amide, was sufficient to attain maximal affinity within the set of native gp41-sequence peptides assayed. Scanning single-residue alanine and d-residue substitutions then confirmed the essential recognition requirements of 2F5 for the central DKW sequence, and also established the importance of the terminal leucine residues in determining high-affinity binding of the linear nonapeptide. Further studies of side-chain and backbone-modified analogs revealed a high degree of structural specificity for the DK sequence in particular, and delineated the steric requirements of the Leu(3) and Trp(6) residues. The nine-residue 2F5 epitope, flanked by pairs of serine residues, retained a high affinity for 2F5 when it was conformationally constrained as a 15-residue, disulfide-bridged loop. However, analogs with smaller or larger loop sizes resulted in lower 2F5 affinities. The conformational effects of the gp41 C-peptide helix immediately adjacent to the N-terminal end of the ELDKWA epitope were examined through the synthesis of helix-initiated analogs. Circular dichroism (CD) studies indicated that the alpha-helical conformation was propagated efficiently into the LELDKWASL epitope, but without any significant effect on its affinity for 2F5. This study should guide the design of a second generation of conformationally constrained ELDKWA analogs that might elicit an immune response that mimics the HIV-neutralizing actions of 2F5.

Structure and Mechanistic Analysis of the Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5 in Complex with Its gp41 Epitope

“…Since then, protease protection, phage display, and peptide binding experiments have shown its epitope to be larger (2,46,59,68), with addition of the two leucines flanking the core epitope increasing the affinity of 2F5 by 2,000-fold (59). The affinity of 2F5 for the membrane-proximal region is also enhanced when gp41 is presented in the presence of lipid (21), suggesting that the idiotope of the membrane-proximal region in vivo may depend on the presence of membrane.…”

“…These include nuclear magnetic resonance (NMR) studies of the membrane-proximal region (2,3) and crystal structures of 2F5 alone or in complex with a core 7-mer peptide (5,44). These studies have generally shown that the core epitope contains a ␤-turn conformation and have subsequently led to the hypothesis that cyclically constrained immunogens might more accurately mimic the 2F5 epitope and thus might more effectively induce 2F5-like neutralizing antibodies (36,59). However, immunogenic studies based on the 7-mer core epitope, using cyclically constrained peptides, have thus far failed to elicit broadly neutralizing antibodies (36).…”

HIV-1 Glycoprotein Immunogenicity

“…However, the role of the presence of lipids in recognition of epitopes during the membrane fusion has not yet been clearly elucidated. The 2F5 epitope contains a -turn conformation and antigen which is presented in a similar context may better mimic the epitope and consequently improve the binding of this Ab (Tian et al, 2002). Several difficulties rise to make a stable immunogen, flexible and mimicking faithfully the MPER in a native conformation sported during the steps of rapprochement and viral fusion process.…”

“…Studies have shown, for expression library and protection against proteases, that this epitope is wider (EQELLELDKWASLWN). By addition of a leucine at each side (LELDKWASL), the affinity for its epitope is increased by more than 2000 times (Ofek et al, 2004;Parker et al, 2001;Tian et al, 2002). In models that study the involvement of the viral membrane using POPC (1-oleyl-2-palmolityl-sn-glycero-3-phosphocholine) and POPC/cholesterol in the recognition and binding to its epitope, it has been shown that 2F5 does not interact with the membrane before fixation (Veiga and Castanho, 2006).…”

Applications of immunochemistry in human health: advances in vaccinology and antibody design (IUPAC Technical Report)

“…an α-aminoisobutyric (Aib) residue], by side-chain cross-linking or "stapling", and the use of helix caps and hydrogen bond surrogates. Some of these approaches have been explored already in vaccine design efforts, for example, the use of Aib residues to favour helical turns, hydrazone crosslinks as hydrogen bond surrogates, Freidinger-like lactams and pseudoprolines to stabilize turns, and cross-linked side-chains to stabilize helical epitopes [158][159][160][161][162][163][164][165]. β-Hairpin mimetics might also be very useful in synthetic vaccine design.…”