Fig. 2: Classification and neutralizing activity of isolated nanobodies against SARS-CoV-2 variants and diverse hACE2-dependent sarbecoviruses.

a Classification of nanobodies into four major groups based on their degrees of competition with ACE2 and control eCR3022 antibody with known epitope specificity, measured by surface plasmon resonance (SPR). “++++” indicates >90% competition, “+++” 60-90%, “++” 30-60%, “+” 10-30%, and “-” no detectable competition. Neutralizing activities of nanobodies in the Fc format against WT D614G were presented by IC50 (μg/mL and nM) while that against SARS-CoV-2 variants and hACE2-dependent sarbecoviruses were in fold-changes relative to that of WT D614G. IC50 values highlighted in salmon indicate <0.02 μg/mL; in yellow 0.02-0.1 μg/mL, and in green >0.1 μg/mL. “-” indicates increased resistance and “+” indicates increased sensitivity to nanobody neutralization. The fold-changes highlighted in light red indicate that resistance increased at least 3-fold; in light blue, sensitivity increased at least threefold; and in white, resistance or sensitivity increased less than 3-fold. BDL (below detection limit) in dark red indicates that nanobodies at their highest concentration (13.33 μg/mL) failed to reach 50% neutralization. Results were calculated from three independent experiments and each performed in technical duplicates. b Neutralizing activity of representative nanobodies from G1, G2, and G3 against authentic SARS-CoV-2 of wildtype (WT) and Alpha, Beta, Delta and Omicron VOCs. Data are presented as mean ± SD. The results shown are representatives of two independent experiments. See also Fig. S2, S3 and Table S1. Source data are provided as a Source Data file.