Hidden complexity of glycan-dependent broadly neutralizing HIV antibodies uncovered by digital panning and native trimer

Precursors and intermediates of HIV-1 broadly neutralizing antibodies (bNAbs) are the key to Bcell-lineage immunogen design. In this study, we analyzed the antibody libraries from donor 17, the source of glycan-dependent PGT121-class bNAbs, using a native, prefusion-optimized trimer probe and the “digital panning” method, which combines 900bp deep sequencing and H/L-paired antibodyomics. In addition to single-chain variable fragments (scFvs) resembling bNAbs, digitalpanning also identified early bNAb intermediates with notable binding affinities for the native trimer and broad neutralization. By examining mouse sera from our previous immunization, we further demonstrated that a scaffolded trimer, without mutations to the glycan shield, could elicit robust responses to the N332 supersite that were blocked by donor-derived bNAb intermediates. Our study thus revealed the hidden lineage complexity of the PGT121 class, which will facilitate future vaccine development targeting the N332 supersite, while digital panning provides a useful new tool for identifying rare bNAb intermediates.