The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naïve human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report 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. Furthermore, 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. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.
Membrane fusion of HIV-1 with its target cells represents the first step in viral infection. This process involves a series of conformational changes in two viral envelope glycoproteins, gp120 and gp41, subsequent to binding of gp120 to the CD4 receptor and the chemokine coreceptor on the target cell membrane. During the fusion process, the conserved N-heptad repeat (N-HR) of gp41 in the form of a trimeric coiled-coil is accessible and presents an attractive target for the generation of broadly neutralizing antibodies. Here we present the crystal structures of two monoclonal Fabs complexed to a mimetic of the N-HR trimer. These Fabs were derived from a synthetic human combinatorial antibody library comprising more than 10 10 human specificities by first panning against an N-HR mimetic, followed by affinity maturation through targeted diversification of the CDR-H2 complementarity determining region. One of the Fabs is broadly neutralizing across a wide range of primary isolates from subtype B and C HIV-1, whereas the other one is non-neutralizing. Our structures reveal the key role of the CDR-H2 loop in antigen recognition and how this correlates with HIV-1 neutralization properties.