The heavy chain and light chain of each Fab were co-transfected into HEK293F cells in a 2:1 ratio (90 μg of DNA total per 200 mL culture) using the FectoPRO transfection reagent (Polyplus Transfections) (1:1 ratio of DNA:FectoPRO) at a cell density of 0.8 × 106 cells mL−1. Cultures were incubated in a Multitron Pro shaker (Infors HT) at 37 °C, 125 rpm, 70% humidity, and 8% CO2. One week later, cells were harvested at 6000×g for 30 min and resulting supernatants were filtered with a 0.22 μm filtration device (EMD Millipore).

Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions.