Ablation of the Complementarity-Determining Region H3 Apex of the Anti-HIV-1 Broadly Neutralizing Antibody 2F5 Abrogates Neutralizing Capacity without Affecting Core Epitope Binding

Abstract

The identification and characterization of broadly neutralizing antibodies (bnAbs) against HIV-1 has formed a major research focus, with the ultimate goal to help in the design of an effective AIDS vaccine. One of these bnAbs, 2F5, has been extensively characterized, and residues at the apex of its unusually long complementaritydetermining region (CDR) H3 loop have been shown to be crucial for neutralization. Structural studies, however, have revealed that the 100 TLFGVPI 100F apex residues of the CDR H3 loop do not interact directly with residues of its core gp41 epitope. In an attempt to gain better insight into the functional role of this element, we have recombinantly expressed native 2F5 Fab and two mutants in which either the apical Phe100B(H) residue was changed to an alanine or the CDR H3 residues 100 TLFGVPI 100F were replaced by a Ser-Gly dipeptide linker. Isothermal titration calorimetry (ITC) and competitive-binding enzyme-linked immunosorbent assays (ELISAs) rendered strikingly similar affinity constants (K d [dissociation constant] of ϳ20 nM) for linear peptide epitope binding by 2F5 Fabs, independent of the presence or absence of the apex residues. Ablation of the CDR H3 apex residues, however, abolished the cell-cell fusion inhibition and pseudovirus neutralization capacities of 2F5 Fab. We report competitive ELISA data that suggest a role of 2F5 CDR H3 apex residues in mediating weak hydrophobic interactions with residues located at the C terminus of the gp41 membrane proximal external region and/or membrane components in the context of core epitope binding. The present data therefore imply an extended 2F5 paratope that includes weak secondary interactions that are crucial for neutralization of Env-mediated fusion.

Enhanced HIV-1 neutralization by antibody heteroligation

“…Although lipid reactivity is required for the neutralizing activity of these antibodies, the mechanism that enhances neutralization is not precisely defined (72,(74)(75)(76). One possibility is that neutralization is enhanced by bivalent heterotypic binding.…”

Elicitation of structure-specific antibodies by epitope scaffolds

“…Nonetheless, we do not expect these antibodies to recreate fully the antibody properties of 2F5, because the membrane-binding component of 2F5 recognition was not addressed in the design procedure. This component is responsible for a substantial portion of the free energy of 2F5 recognition, without which neutralization is difficult to attain (15,16,29,30). A number of other groups have also studied the expression of the nominal 2F5-epitope sequence (ELDKWAS) in scaffold systems: a variable loop of the HIV-1 gp120 envelope glycoprotein; a surface loop of human rhinovirus; and a surface loop of bovine papilloma virus (25,(31)(32)(33).…”

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

“…These concepts can be usefully defined in more than one way (15), but for some purposes, both terms can be specified precisely and quantitatively by reference to thermodynamics. However, very few data have been reported to date about the thermodynamics of binding of the interaction between bNAb 2F5 and its corresponding epitope peptide, and they account only for the Fab fraction (16). In this study, we have characterized thermodynamically the binding of bNAb 2F5 to peptides corresponding to both the core and functional epitopes by isothermal titration calorimetry (ITC).…”