Characterization of the steric defense of the HIV‐1 gp41 N‐trimer region

During viral entry, HIV gp41 adopts a transient conformation called the “prehairpin intermediate” in which a highly conserved therapeutic target, the N‐trimer, is exposed. Despite extensive discovery efforts, potent and broadly neutralizing antibodies that target the N‐trimer are elusive. We previously demonstrated the N‐trimer is protected by a steric block that prevents large proteins, such as antibodies, from accessing it. Here we further characterize the steric block and identify its source. To study the N‐trimer steric accessibility, we produced two sets of C‐peptide inhibitors (a potent inhibitor targeting the N‐trimer) fused to cargo proteins of increasing size facing either the virus or cell side of the prehairpin intermediate. Both bulky inhibitor sets show a steric block, but the effect is more pronounced with virus‐side cargo. Additionally, both sets maintain their potencies in a modified entry assay that removes possible sources of target cell steric hindrance. These results implicate a viral source, likely gp120, as the primary component of the steric block. In addition, we studied the steric accessibility of the “pocket” region of the N‐trimer, a highly attractive drug and vaccine target. We demonstrated a pocket‐specific antibody, D5, is more potent as an scFv than as a full‐length IgG, suggesting the N‐trimer steric restriction extends to the pocket. This characterization will facilitate the design of sterically restricted antigens that mimic the steric environment of the N‐trimer in the prehairpin intermediate and are capable of inducing potent and broadly neutralizing antibodies that circumvent the N‐trimer steric block.