Fig. 2: Potently neutralizing VHHs bind to three non-overlapping antigenic sites.

a Half-maximal inhibitory concentration (IC₅₀) values for 16 potently neutralizing VHH candidates determined by neutralization assays performed with recombinant RV3-eGFP virus. Points represent geometric means of biological replicates (n = 2 for 2B03, 3A02, 3E06, 4E07, 5D03, 5E03, 6B02, and 6B03; n = 3 for 1A03, 1H09, 2E10, 2H02, 4C03, and 4C06; n = 4 for 1D10 and 3B04) ±geometric standard deviations. Representative curves are displayed in Supplementary Fig. 3 and numeric IC₅₀ values are listed in Supplementary Table 1. b ELISA binding curves for the top 13 VHH candidates. VHHs were tested for binding to RV3 preF (left) and postF (right). Half maximal effective concentration (EC₅₀) values are listed in Supplementary Table 3. c Competition matrix indicating the level of binding observed for each of the 13 top VHH candidates to preF saturated with the same VHH or a different VHH from the same pool. Columns indicate the competition profile of each VHH. Each epitope bin is outlined based on the representative VHH that was selected for further characterization, colored as in a and b. d Sequence alignment of the representative VHH selected from each epitope bin. VHHs 4C06, 1H09, and 1D10 are aligned to the 4C03 sequence. IMGT-based residue numbers are represented by blue text. Invariant residues are indicated by black dots. Complementarity determining regions (CDRs) are indicated by boxes. e Surface plasmon resonance sensorgrams for binding of each representative VHH to preF. Binding curves are colored black. Data fit to a 1:1 binding model is colored by VHH according to a and b. Source data are provided within the Source Data file.