Structural Characterization of a Neutralizing Nanobody With Broad Activity Against SARS-CoV-2 Variants

SARS-CoV-2 and its variants, such as the Omicron continue to threaten public health. The virus recognizes the host cell by attaching its Spike (S) receptor-binding domain (RBD) to the host receptor, ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here, we report the isolation and biological and structural characterization of a single-chain antibody (nanobody) from RBD-immunized alpaca. The nanobody, named DL28, binds to RBD tightly with a K(D) of 1.56 nM and neutralizes the original SARS-CoV-2 strain with an IC(50) of 0.41 μg mL(−1). Neutralization assays with a panel of variants of concern (VOCs) reveal its wide-spectrum activity with IC(50) values ranging from 0.35 to 1.66 μg mL(−1) for the Alpha/Beta/Gamma/Delta and an IC(50) of 0.66 μg mL(−1) for the currently prevalent Omicron. Competition binding assays show that DL28 blocks ACE2-binding. However, structural characterizations and mutagenesis suggest that unlike most antibodies, the blockage by DL28 does not involve direct competition or steric hindrance. Rather, DL28 may use a “conformation competition” mechanism where it excludes ACE2 by keeping an RBD loop in a conformation incompatible with ACE2-binding.