Background:  Using phage display and synthetic peptides that mimic a conformational intermediate of gp41 formed during viral fusion, we have identified a novel HIV-neutralizing monoclonal antibody termed D5. The peptides (5-Helix and IZN36) share the Heptad Repeat 1 (HR1) region of the gp41 ectodomain that forms a hydrophobic groove which is the target of the anti-viral peptide T20. During viral fusion, the HR1 groove interacts with the helical HR2 segments of gp41 to form a trimeric hairpin structure or 6-Helical bundle, enabling juxtaposition of viral and cellular membranes. Like T20, D5 prevents formation of a 6-helical bundle in vitro, which likely underlies D5’s anti-viral activity.

Methods:  We now report that D5’s epitope is located in a previously well-characterized hydrophobic pocket present in the HR1 groove that normally accommodates 3 key residues (W628, W631, and I635) from the N-terminal portion of the HR2 region during formation of the hairpin structure. This pocket is also the target of anti-viral cyclic D-peptides that were identified by mirror-image phage display. We have mapped the precise amino acids of this pocket that are required for D5 binding and find that they are highly conserved in HIV-1.

Results:  Three amino acids (L568, W571, and K574) are absolutely essential for binding, while an additional residue (V570) contributes to binding but is not essential. NMR spectroscopy confirmed that the residues participating in D5 binding are situated in the HR1 hydrophobic pocket. When individual point mutations corresponding to the essential amino acids comprising the D5 epitope were introduced into a molecular clone of HIV-1, resulting viruses were either compromised with regard to infectivity (L568A and K574A), or were rendered non-infectious (W571A). Testing of the two infectious viruses for susceptibility to D5 revealed that although both viruses were less sensitive to inhibition by D5, remarkably, both showed enhanced susceptibility to other fusion inhibitors such as T20.

Results:  Collectively, these new findings reveal that D5 interacts with a highly conserved epitope located in the hydrophobic pocket formed by the gp41 HR1 region. Future studies will be aimed at exploiting this novel conserved epitope in vaccination strategies by attempting to elicit polyclonal D5-like antibodies in experimental animals using synthetic peptides containing the HR1 hydrophobic pocket.

Keywords: gp41; heptad-repeat-1; Neutralizing antibody