A Derivative of the D5 Monoclonal Antibody That Targets the gp41 N-Heptad Repeat of HIV-1 with Broad Tier-2-Neutralizing Activity.

HIV-1 infection is initiated by the viral glycoprotein Env, which, after interaction with cellular coreceptors, adopts a transient conformation known as the prehairpin intermediate (PHI). The N-heptad repeat (NHR) is a highly conserved region of gp41 exposed in the PHI; it is the target of the FDA-approved drug enfuvirtide and of neutralizing monoclonal antibodies (mAbs). However, to date, these mAbs have only been weakly effective against tier-1 HIV-1 strains, which are most sensitive to neutralizing antibodies. Here, we engineered and tested 11 IgG variants of D5, an anti-NHR mAb, by recombining previously described mutations in four of D5's six antibody complementarity-determining regions. One variant, D5&#95;AR, demonstrated 6-fold enhancement in the 50% inhibitory dose (ID ₅₀ ) against lentivirus pseudotyped with HXB2 Env. D5&#95;AR exhibited weak cross-clade neutralizing activity against a diverse set of tier-2 HIV-1 viruses, which are less sensitive to neutralizing antibodies than tier-1 viruses and are the target of current antibody-based vaccine efforts. In addition, the neutralization potency of D5&#95;AR IgG was greatly enhanced in target cells expressing FcγRI, with ID ₅₀ values of <0.1 μg/ml; this immunoglobulin receptor is expressed on macrophages and dendritic cells, which are implicated in the early stages of HIV-1 infection of mucosal surfaces. D5 and D5&#95;AR have equivalent neutralization potency in IgG, Fab, and single-chain variable-fragment (scFv) formats, indicating that neutralization is not impacted by steric hindrance. Taken together, these results provide support for vaccine strategies that target the PHI by eliciting antibodies against the gp41 NHR and support investigation of anti-NHR mAbs in nonhuman primate passive immunization studies. IMPORTANCE Despite advances in antiretroviral therapy, HIV remains a global epidemic and has claimed more than 32 million lives. Accordingly, developing an effective HIV vaccine remains an urgent public health need. The gp41 N-heptad repeat (NHR) of the HIV-1 prehairpin intermediate (PHI) is highly conserved (>90%) and is inhibited by the FDA-approved drug enfuvirtide, making it an attractive vaccine target. However, to date, anti-NHR antibodies have not been potent. Here, we engineered D5&#95;AR, a more potent variant of the anti-NHR antibody D5, and established its ability to inhibit HIV-1 strains that are more difficult to neutralize and are more representative of circulating strains (tier-2 strains). The neutralizing activity of D5&#95;AR was greatly potentiated in cells expressing FcγRI; FcγRI is expressed on cells that are implicated at the earliest stages of sexual HIV-1 transmission. Taken together, these results bolster efforts to target the gp41 NHR and the PHI for vaccine development.