Research is focused on studying the events involved in HIV-1 fusion, the development of fusion inhibitors directed against gp41 and the development of monoclonal antibodies with broadly neutralizing activity that are directed against gp41. The HIV-1 Envelope (Env) proteins that mediate membrane fusion represent a major target for the development of new AIDS therapies. Three classes of Env-mediated membrane fusion inhibitors have been described that specifically target the pre-hairpin intermediate conformation of gp41. Class 2 inhibitors bind to the C-terminal heptad repeat (C-HR) of gp41. The single example of a class 3 inhibitor targets the trimeric N-terminal heptad repeat (N-HR) of gp41 and has been postulated to sequestrate the N-HR of the pre-hairpin intermediate through the formation of fusion incompetent heterotrimers. We have now shown that NCCG-gp41, a class 2 inhibitor, and N36Mut(e,g), a class 3 inhibitor, synergistically inhibit Env-mediated membrane fusion for several representative HIV-1 strains (X4 and R5) in both a cell fusion assay (with membrane-bound CD4) and an Env-pseudotyped virus neutralization assay. We have also succeeded in obtaining a monoclonal Fab (Fab 3674) selected from a human non-immune phage library by panning against the chimeric construct NCCG-gp41 (that comprises an exposed coiled-coil trimer of gp41 N-helices fused in helical phase onto the minimal thermostable ectodomain of gp41) that effectively neutralizes diverse laboratory-adapted B-strains of HIV-1 and primary isolates of subtypes A, B and C in an Env-pseudotyped virus neutralization assay, albeit with reduced potency (25x) compared to 2F5 and 4E10. Alanine scanning mutagenesis maps a novel epitope to a shallow groove on the N-helices of gp41 that is exposed between two C-helices in the fusogenic six-helix bundle conformation of gp41. We have shown that bivalent Fab 3674 and the C34 peptide (a potent fusion inhibitor derived from the C-helix of gp41) act at similar stages of the fusion reaction and to neutralize HIV-1 synergistically, providing additional evidence that the epitope of Fab 3674 is new and distinct from the binding site of C34.More recently we have shown that the class 3 inhibitor N36Mut(e,g) prolongs the temporal window during which the virus is susceptible to neutralization by the bivalent Fab-3674, and that bivalent Fab-3674 and N36Mut(e,g) neutralize HXB2 and SF-162 strains of HIV-1, as well as diverse primary B and C clade HIV-1 strains, synergistically in a Env-pseudotyped virus neutralization assay. N36Mut(e,g) also rescues neutralizing activity of the monovalent Fab-3674 against resistant HIV-1 strains and renders a series of related non-neutralizing Fabs neutralizing. Moreover, N36Mut(e,g) exhibits the same effects on the broadly neutralizing 2F5 and 4E10 monoclonal antibodies directed against the membrane proximal extended region of gp41. Very recently, we have subjected Fab 3674 to affinity maturation against the NCCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtypes B and C HIV-1 reference panels. The parental Fab 3674 is 10-20 fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (120 kDa) across the subtypes B and C reference panels. We have now solved crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5 (developed by Merck), derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop. A key feature of fusion inhibitors that target the N-HR trimer, including antibodies directed against the N-HR trimer, is that deactivation of gp41 in vivo is a slow reversible process that is dependent on chemokine receptor binding to Env, and that the exposed N-HR trimer remains accessible to inhibitors until the final conformational changes in gp41 that lead to the formation of the 6-HB have taken place. This suggests that inhibition of fusion will be most effective when two or more Fabs are bound to the exposed N-HR trimer of the pre-hairpin intermediate of gp41. Since multiple Fabs/antibodies bound to the N-HR trimer are unlikely to dissociate simultaneously, the probability that at least one antibody is bound to the N-HR trimer at all times will be increased. Modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Thus, in the case of the complex with the most potent neutralizing Fab in our series, Fab 8066, the molecules of Fab can readily bind to the N-HR trimer without any steric clashes between adjacent Fab molecules. In contrast, as a consequence of the different mode of binding of the CDR-H2 to the hydrophobic pocket on the surface of the N-HR trimer, binding of three molecules of Fab 8062 to the N-HR trimer may result in steric clash between adjacent Fab molecules involving the CDR-H2, CDR-L1, CDR-L3 loops and loop 71-78. It seems likely in the light of the current structural data, modeling results and neutralization properties of our Fab series, that neutralization is dependent not only on tight binding of a single Fab to the N-HR trimer but also on the ability to bind multiple Fabs to a single N-HR trimer at preliminary step of fusion process.
