Single-antibody mutational scanning reveals poly-specificity and immune constraints in vaccination responses

Current repertoire technologies with single-antibody resolution usually assess binding to only one or a few recombinant antigens, neglecting variant diversity of antigens and poly-specificity of antibodies. Here, we introduce a method for analyzing antibody repertoires at single-antibody resolution against a large library of SARS-CoV-2 receptor-binding domain (RBD) variants, enabling the identification and analysis of recognized RBD variants by rare, poly-specific antibodies. This technique provided unique insights into single-antibody binding and escape profiles within a murine immunization model with different RBDs, offering valuable data useful to optimize vaccine design against emerging variants and study antibody poly-specificity.