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.

The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the membrane-proximal external region (MPER) that connects the human immunodeficiency virus type 1 (HIV-1) envelope gp41 ectodomain with the transmembrane anchor. By adopting different conformations that stably insert into the virion external membrane interface, such as helical structures, a conserved aromatic-rich sequence within the MPER is thought to participate in HIV-1-cell fusion. Recent experimental evidence suggests that the neutralizing activity of 2F5 and 4E10 might correlate with the MAbs' capacity to recognize epitopes inserted into the viral membrane, thereby impairing MPER fusogenic activity. To gain new insights into the molecular mechanism underlying viral neutralization by these antibodies, we have compared the capacities of 2F5 and 4E10 to block the membrane-disorganizing activity of MPER peptides inserted into the surface bilayer of solution-diffusing unilamellar vesicles. Both MAbs inhibited leakage of vesicular aqueous contents (membrane permeabilization) and intervesicular lipid mixing (membrane fusion) promoted by MPER-derived peptides. Thus, our data support the idea that antibody binding to a membrane-inserted epitope may interfere with the function of the MPER during gp41-induced fusion. Antibody insertion into a cholesterol-containing, uncharged virion-like membrane is mediated by specific epitope recognition, and moreover, partitioning-coupled folding into a helix reduces the efficiency of 2F5 MAb binding to its epitope in the membrane. We conclude that the capacity to interfere with the membrane activity of conserved MPER sequences is best correlated with the broad neutralization of the 4E10 MAb.It is generally accepted that a potentially successful human immunodeficiency virus (HIV) vaccine should be capable of eliciting a robust neutralizing antibody (NAb) response (15, 16). The isolation from infected asymptomatic individuals of several antibodies showing neutralizing activity against a broad spectrum of viral clades implies that triggering such an immunogenic response might conceivably be possible (15,16,74). The anti-gp41 2F5 and 4E10 monoclonal antibodies (MAbs) constitute a paradigm in this respect (21,34,48,49,56,68). These MAbs simultaneously exert broad and potent neutralizing activity across different HIV type 1 (HIV-1) strains and primary isolates (9,34,45,48,68,89), confer protection against viral infection when passively transferred to primate models (67), and increase the neutralization response upon passive immunization of humans (66,74). Thus, deciphering the molecular mechanisms underlying viral cross-neutralization by 2F5 and 4E10 poses a challenge for researchers working on HIV vaccine development (86).2F5 and 4E10 recognize conserved linear epitopes located within the membrane-proximal external region (MPER) of the HIV-1 gp41 fusogenic Env subunit (7,18,19,34,48,49,51,54,89). The crystal structures of the 2F5 and 4E10 Fabs in a complex with soluble epitope derivatives hav...

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