Crystal Structure and Size-Dependent Neutralization Properties of HK20, a Human Monoclonal Antibody Binding to the Highly Conserved Heptad Repeat 1 of gp41

Abstract

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool. The HIV-1 envelope glycoprotein composed of the receptor binding subunit gp120 and the fusion protein gp41 is the prime target for neutralizing antibodies. Receptor binding induces a conformational change in gp41 that transiently exposes the conserved heptad repeat 1 (HR1) region. We have previously isolated the human HR1-specific mAb HK20 and provide now the structural basis for epitope recognition. HK20 employs mainly its CDR H2 and H3 for binding similar to HR1 binding of mAb D5. We demonstrate that HK20 and D5 bind HR1 with similar affinities; however, HK20 has a broader neutralization breadth than D5, which might be due to the differences in their approach angles of epitope recognition. Competition analyses of 33 sera from HIV-1 infected individuals reveal significant titers of HK20-inhibiting antibodies in 20 cases, confirming the immunogenicity of the epitope. We demonstrate further that HK20 IgG have limited neutralization breadth and potency while smaller HK20 Fabs and scFv reveal a broad cross clade neutralization breadth. This suggests that the accessibility of the HR1 epitope limits the value of HR1 mAbs for infection prevention, but highlights the importance of smaller versions such Fabs or scFv to combat infection alone or in synergistic approaches with other antivirals.