A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope

Abstract

HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H͞I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusioninhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a ''hot spot'' for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.envelope ͉ fusion ͉ prehairpin ͉ vaccine

Pharmacological applications of a novel neoepitope antibody to a modified amyloid precursor protein-derived beta-secretase product

“…Selection of phage displayed human scFv antibodies specific to EV neoepitope from the CAT libraries To select EV neoepitope-specific scFv phage, solution based panning was performed using three pooled CAT libraries (BMV, CS and DP47) (Miller et al, 2005;An et al, 2009;Montgomery et al, 2009). The libraries were initially subtracted for phages that bind to the NFEV substrate peptide, followed by panning against EV peptide (EVEFRHDSGYK-biotin) coated magnetic beads (Table 1).…”

“…its stability and affinity for the EV neoepitope peptide (Miller et al, 2005;An et al, 2009;Montgomery et al, 2009;Palys et al, 2006). scFv heavy (H) and light (L) chain cDNA was cloned into human IgG1 expression vectors PEU1.2 and PEU4.2, respectively (Miller et al, 2005;Montgomery et al, 2009).…”

“…scFv heavy (H) and light (L) chain cDNA was cloned into human IgG1 expression vectors PEU1.2 and PEU4.2, respectively (Miller et al, 2005;Montgomery et al, 2009). The H and L chain DNA plasmids were co-transfected transiently into the HEK293-EBNA cell line and the purified EV3 IgG antibody was characterized for purity and integrity.…”

“…Three phage displayed scFv libraries (BMV, CS and DP-47) were obtained from Cambridge Antibody Technologies (CAT, Cambridge, United Kingdom) (Miller et al, 2005;An et al, 2009;Montgomery et al, 2009). The pooled libraries were panned in a 3-step procedure based on methods described (Vaughan et al, 1996).…”

“…scFv phage clones specific to peptide with N-terminal amino acid E were selected for DNA extraction and sequencing of both the scFv frame region (FR) and CDRs using the sequencing oligonucleotide primers from CAT as previously described (Miller et al, 2005;An et al, 2009;Montgomery et al, 2009). …”