Strain-dependent neutralization reveals antigenic variation of human parechovirus 3

Figure 1. Neutralization capacity of a polyclonal rabbit HPeV3 antiserum (HPeV3 pAb), the Dutch (NL) and the Japanese (JP) IVIG batches and a human AT12-015 monoclonal antibody (HPeV3 mAb) against a panel of 25 HPeV3 isolates. (a) Heat map representation of log2-transformed nAb titers against individual HPeV3 isolates arranged based on year of isolation. (b) Comparison of prototype HPeV3 A308/99 strain nAb titers against the median nAb titers (with interquartile range) of the clinical HPeV3 isolates. Samples were grouped by the geographical location of isolation (Dutch isolates; NL, Japanese isolates; JP and Australian isolates; AUS). Positivity was defined as a titer ≥5 log2. HPeV3 isolate group median nAb titers with interquartile range were compared by Kruskal-Wallis test with Dunn’s post hoc analysis (significance level p < 0.05).

Figure 2. Amino acid variation in the capsid-encoding region of a subset of HPeV3 isolates. (a) Alignment showing the positions of variable amino acid residues. Capsid surface-exposed residues are highlighted in red. (b) HPeV3 roadmap of capsid exterior. Five full asymmetric units are shown; each formed of one copy of capsid proteins VP0 (blue), VP3 (green) and VP1 (pink). The AT12-015 Fab footprint (yellow contour) is mapped on surface residues differing between the strains (red), in comparison to the prototype A308/99. Residue-labeled close-up roadmaps for each strain are showed in Supplementary Information 1.

Figure 3. Characterization of the AT12-015 mAb resistant (MAR) HPeV3 variant. (a) Nonlinear regression analysis of the neutralizing activity of a human AT12-015 mAb against the HPeV3 A308/99 wild-type (wt) and the MAR HPeV3 variant. Data presented as the mean ± SD. (b) Immunofluorescence imaging of HPeV3 A308/99 wt and MAR variant infected HT29 cells stained by a rabbit HPeV1 antiserum (HPeV1 pAb) or the AT12-015 mAb. (c) Amino acid mutations in the MAR HPeV3 variant as compared to the A308/99 wt and the clinical isolates. Capsid surface-exposed residues are highlighted in red. (d) HPeV3 roadmap of capsid exterior. Capsid proteins VP0 (blue), VP3 (green) and VP1 (pink), the AT12-015 Fab footprint (yellow contour), and also amino acid mutations in variant resistant to AT12-015 are shown (red).