AntibodY used for neutralization assays were produced in Expi293F cells using FectoPRO. VH and VL plasmids were cotransfected at a 1:1 ratio and cells were transfected at 3E+6 cells/mL. Cell cultures were incubated at 37 °C and 8% CO2 with shaking at 120 rpm and harvested 3 days post-transfection by spinning at >4200g for 15 min and then filtered through a 0.45 μm filter.

The N-heptad repeat (NHR) of the HIV-1 gp41 prehairpin intermediate (PHI) is an attractive potential vaccine target with high sequence conservation across diverse strains. However, despite the potency of NHR-targeting peptides and clinical efficacy of the NHR-targeting entry inhibitor enfuvirtide, no potently neutralizing NHR-directed monoclonal antibodies (mAbs) nor antisera have been identified or elicited to date. The lack of potent NHR-binding mAbs both dampens enthusiasm for vaccine development efforts at this target and presents a barrier to performing passive immunization experiments with NHR-targeting antibodies. To address this challenge, we previously developed an improved variant of the NHR-directed mAb D5, called D5_AR, which is capable of neutralizing diverse tier-2 viruses. Building on that work, here we present the 2.7Å-crystal structure of D5_AR bound to NHR mimetic peptide IQN17. We then utilize protein language models and supervised machine learning to generate small (n < 100) libraries of D5_AR variants that are subsequently screened for improved neutralization potency. We identify a variant with 5-fold improved neutralization potency, D5_FI, which is the most potent NHR-directed monoclonal antibody characterized to date and exhibits broad neutralization of tier-2 and -3 pseudoviruses as well as replicating R5 and X4 challenge strains. Additionally, our work highlights the ability of protein language models to efficiently identify improved mAb variants from relatively small libraries.