Nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants

We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted—most successfully, vaccination1–3—imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies)4 have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies5, 6. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)—namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays7. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.