Identification of Human Norovirus GII.3 Blockade Antibody Epitopes

Figures

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  Figure 1. Binding affinity and specificity of the mAbs to GII.3 VLP. (A,B) Biolayer interferometry sensorgrams of anti-GII.3 mAbs 8C7 (A) and 8D1 (B) binding to biotinylated GII.3-KJ VLP on streptavidin-coated biosensors. Association and dissociation steps are divided by dashed lines. The mAb concentrations were shown. (C–F) Reactivity of anti-GII.3 mAbs (8C7, 8D1, 3A3, and 9B8) with GII.3-KJ VLP (C), GI.1 VLP (D), GII.4 VLP (E), and GII.17 VLP (F) were determined by ELISA. Data are mean ± SD of triplicate wells. 7D8, a norovirus cross-reactive antibody, used as a positive control. 2H2, an anti-SARS-CoV-2 mAb, used as a negative control. Conc., the abbreviation for concentration.

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  Figure 2. The ability of the mAbs to block the interaction between GII.3-KJ VLP and HBGA-containing PGM III was tested by ELISA. HBsAg-specific mAb 2G9 was used as a control (ctr). Blocking index (%) was determined by comparing the results of the VLP/MAb wells with the VLP-only wells. Data are mean ± SD of triplicate wells.

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  Figure 3. Cross-reactivity of the 8C7 and 8D1 mAbs was tested by ELISA with various wild-type GII.3 VLPs. Rabbit anti-GII.3 VLP antiserum was used as a positive control. Dashed line indicates the value from the blank well (containing no antigen). Data are mean ± SD of triplicate wells.

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  Figure 4. Epitope mapping of mAbs 8C7 and 8D1. (A) Diagram of the parental and chimeric GII.3 VLPs. The summary of the VLP-binding ELISA results is shown on the right-hand side. +, positive result; –, negative result. (B) Binding activities of the 8C7 and 8D1 mAbs to the indicated parental and chimeric GII.3 VLPs were tested by ELISA. For a given VLP sample, its reactivity with 8C7 or 8D1 was normalized against that with rabbit polyclonal antibody raised against GII.3 VLP, and relative binding was calculated as follows: (OD450 value obtained with mAb—OD450 value of blank well)/(OD450 value obtained with anti-GII.3 polyclonal antibody—OD450 value of blank well). Data are mean ± SEM of triplicate samples.

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  Figure 5. Alignment of the corresponding 8C7 and 8D1 epitope sequences from representative strains of different GII.3 clusters. Information for each strain, including isolation date (year), isolation location (country), and GenBank ID, were shown. The 8C7 and 8D1 epitope regions were boxed with dash lines. Dots represent residues identical to those of the GII.3-KJ strain. Red circle indicates the strains used in this study.

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  Figure 6. The 8C7 and 8D1 epitopes were mapped onto the surface of the GII.3 P domain dimer (strain TV-24; PDB: 6IR5). (A) Model of GII.3 P domain showing the HBGA binding site (green). (B) Location of 8C7 epitope (hot pink) on GII.3 P domain surface. Residues 386 and 388 are shown in red. (C,D) Location of 8D1 epitope (yellow) on GII.3 P domain surface. Panel (A–C), top view; panel (D), side view. The corresponding residues in the HBGA binding site or the mAb epitopes are included in the parentheses.