Structure-guided Alterations of the gp41-directed HIV-1 Broadly Neutralizing Antibody 2F5 Reveal New Properties Regarding its Neutralizing Function

The broadly neutralizing HIV-1 antibody 2F5 recognizes an epitope in the gp41 membrane proximal external region (MPER). The MPER adopts a helical conformation as free peptide, as post-fusogenic forms of gp41, and when bound to the 4E10 monoclonal antibody (Mab). However, when bound to 2F5, the epitope is an extended-loop. The antibody-peptide structure reveals binding between the heavy and light chains with most the long, hydrophobic CDRH3 not contacting peptide. However, mutagenesis identifies this loop as critical for binding, neutralization and for putative hydrophobic membrane interactions. Here, we examined length requirements of the 2F5 CDRH3 and plasticity regarding binding and neutralization. We generated 2F5 variants possessing either longer or shorter CDRH3s and assessed function. The CDRH3 tolerated elongations and reductions up to four residues, displaying a range of binding affinities and retaining some neutralizing capacity. 2F5 antibody variants selective recognition of conformationally distinctive MPER probes suggests a new role for the CDRH3 loop in destabilizing the helical MPER. Binding and neutralization were enhanced by targeted tryptophan substitutions recapitulating fully the activities of the wild-type 2F5 antibody in a shorter CDRH3 variant. MPER alanine scanning revealed binding contacts of this variant downstream of the 2F5 core epitope, into the 4E10 epitope region. This variant displayed increased reactivity to cardiolipin-beta-2-glycoprotein. Tyrosine replacements maintained neutralization while eliminating cardiolipin-beta-2-glycoprotein interaction. The data suggest a new mechanism of action, important for vaccine design, in which the 2F5 CDRH3 contacts and destabilizes the MPER helix downstream of its core epitope to allow induction of the extended-loop conformation. Author Summary: Host antibodies raised in response to acute viral infection are often protective to second exposure. However, in the less frequent examples of chronic infection, in which the virus actively replicates for prolonged periods, host immunity can impact on viral characteristics by applying selective pressures upon progeny. Such a dynamic process is exemplified by the extremely variable and pathogenic human immunodeficiency virus type 1 (HIV-1). Relatively infrequently, antibodies are elicited during infection that can neutralize a diverse array of this malleable pathogen. Hence, studies which elucidate such antibodies are elevated in importance if the pathogen causes human suffering, yet no vaccine exists. Here, we describe a new property of the broadly neutralizing antibody, 2F5, which is directed to a conserved region of the HIV-1 surface protein near the lipid membrane. Through mutagenesis of the antibody and subsequent functional analysis, we present data that suggest a model in which the antibody first binds downstream of its known core epitope in a two-step process not directly involving the lipid membrane. Such studies may better elucidate the not yet defined details of virus-to-cell fusion by which viral DNA enters host target cells. Additionally, such analysis reveals 2F5 binding specificities, important for future vaccine designs.