Fig. 1: Precision library generation and yeast display screening were applied to the template antibody VRC34.01 to enhance recognition of HIV-1 Envelope (Env)-displayed fusion peptide (FP).

A Workflow for precision anti-HIV-1 antibody engineering via yeast display. The VRC34.01 template antibody variable region genes were mutated and selected for improved HIV-1 affinity via successive rounds of site-saturated mutagenesis (SSM) and DNA shuffling. Antibody mutant libraries were screened using FACS to fractionate mutant populations by antigen binding affinity phenotypes. Next-generation sequencing (NGS) was used to mine antibody sequences and bin antibody variants for trimer binding function based on quantitative variant prevalence analysis across sort groups. Biophysical characterization of engineered single- and multi-mutation antibody variants revealed anti-FP antibody sequence-structure-function relationships and defined potent gain-of-function mechanisms that enhanced FP-targeted HIV-1 neutralization. B Yeast libraries expressing antibody in a surface-bound fragment antigen binding (Fab) format were stained with fluorescence markers to measure Fab-surface expression (Y-axis) versus antigen binding (X-axis) and screened via FACS. Template VRC34.01 (top row) and single-mutant amino-acid substitution libraries were generated via SSM across the entire VRC34.01 variable region heavy (VH) and variable region light (VL) genes (middle row: pre-sorting populations; bottom row: round 3 high-affinity enriched populations). Libraries are shown bound to diverse FP sequences displayed on HIV-1 trimer probes. Single-mutant libraries were fractionated in three sequential rounds into high-, medium-, and low-affinity performance bins using FACS, resulting in sorted libraries with phenotypically observable differences in trimer binding. Medium-, and low-affinity plots are also provided in Supplemental Fig. 1A.