ABSTRACT
Vaccine boosters and infection can facilitate the development of SARS-CoV-2 antibodies with improved potency and breadth. Here, we observed super-immunity in a camelid extensively immunized with the SARS-CoV-2 receptor-binding domain (RBD). We rapidly isolated a large repertoire of specific ultrahigh-affinity nanobodies that bind strongly to all known sarbecovirus clades using integrative proteomics. These pan-sarbecovirus nanobodies (psNbs) are highly effective against SARS-CoV and SARS-CoV-2 variants including the Omicron, with the best median neutralization potency at single-digit ng/ml. Structural determinations of 13 psNbs with the SARS-CoV-2 spike or RBD revealed five epitope classes, providing insights into the mechanisms and evolution of their broad activities. The highly evolved psNbs target small, flat, and flexible epitopes that contain over 75% of conserved RBD surface residues. Their potencies are strongly and negatively correlated with the distance of the epitopes to the receptor binding sites. A highly potent, inhalable and bispecific psNb (PiN-31) was developed. Our findings inform on the development of broadly protective vaccines and therapeutics.