Fig 2. Non-RBD epitopes enhance the cell entry of prototypic SARS-CoV-2 pseudoviruses.

Retroviruses pseudotyped with prototypic SARS-CoV-2 spike (i.e., prototypic SARS-CoV-2 pseudoviruses) entered ACE2-expressing cells in the presence of one of the nanobodies at various concentrations. Entry efficiency was characterized as the luciferase signal accompanying entry. The efficacy of each nanobody in neutralizing pseudovirus entry was expressed as the concentration capable of neutralizing pseudovirus entry by 50% (i.e., IC₅₀). (A) Activities of three Fc-tagged RBD-targeting nanobodies: they all potently neutralized SARS-CoV-2 pseudovirus entry. Error bars represent SEM (n = 3). (B) Activities of three Fc-tagged non-RBD-targeting nanobodies: Nanosota-5-Fc enhances pseudovirus entry, Nanosota-6-Fc enhances pseudovirus entry at low concentrations and neutralizes pseudovirus entry at high concentrations, and Nanosota-7-Fc neutralizes pseudovirus entry at high concentrations. Error bars represent SEM (n = 4). (C) Activities of three His-tagged non-RBD-targeting nanobodies: Nanosota-5-His and Nanosota-6-His enhance pseudovirus entry using an FcR-independent mechanism. Error bars represent SEM (n = 4). (D) Summary of the activities of the six spike-binding nanobodies. NA: not available.