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.

Role of Human Immunodeficiency Virus Type 1 Envelope Structure in the Induction of Broadly Neutralizing Antibodies

“…These aspects influence the potency and breadth of neutralization, which are higher for HK20 than for D5 and depend on somatically mutated residues. In addition, it was shown that in the case of HK20, the scFv is at least 15-fold more potent in neutralization than IgG, which is consistent with a limited accessibility to the target site (112). The gp41 footprints of HK20 and D5 and the global structural principles employed by the two antibodies are similar.…”

“…The gp41 footprints of HK20 and D5 and the global structural principles employed by the two antibodies are similar. Since HK20 targets HR1 instead of MPER or glycans in this region, it has the conceptual advantage over 4E10 and 2F5 of avoiding potential autoreactivity (112). HK20 presents an intermediate breadth of neutralization, preferentially to clades A and C, which did not correspond with the subtype of the infected patient, which was CRF02_AG as described in reference 1.…”

Particle Conformation Regulates Antibody Access to a Conserved GII.4 Norovirus Blockade Epitope

“…To estimate the fraction of antibodies specific for conserved GII.4 epitopes in the overall serum antibody response, 100 serum samples collected from healthy individuals were assayed for the ability to block binding of human MAbs NVB 61.3 and NVB 71.4 in a BOB assay (57,58). Both human MAbs recognize a broad panel of antigenically diverse, epidemiologically significant GII.4 NoV strain VLPs by EIA, but only NVB 71.4 blocks VLP-ligand interactions (47).…”

Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

“…Fig. 4A shows a superposition of the covNHR3-ABC structure to the theoretical model of the gp41 ectodomain generated from the coordinates of the simian immunodeficiency virus gp41 ectodomain (1IF3) (21) and the crystallographic structure of the five-helix construct (2XRA) (7). The backbones of the two parallel NHR helices are wellsuperimposed, with an rmsd of 1.4 Å.…”

“…This exposed conformation was shown by the elicitation of Abs that block HIV fusion using immunogens exposing an NHR coiled coil (5). Also, two human mAbs (D5 and Hk20) recognize a highly conserved NHR hydrophobic pocket and neutralize diverse HIV-1 clinical isolates (6,7). Moreover, potent HIV-1-neutralizing CHR peptides, pioneered by T20, and small molecules bind to the trimeric NHR, interfering with formation of the NHR/CHR six-helix bundle (8)(9)(10)(11)(12).…”