Fig 1.
Characterization of IgG1 and IgM mAbs from EV-A71 infected children.
(A) numbers and percentages of EV-A71 neutralizing IgG1 mAbs, non-neutralizing EV-A71 binding IgG1 mAbs and non-binding IgG1 mAbs. (B) EV-A71 binding activity of 56 IgG1 mAbs from the five study subjects were determined using ELISA. (C) numbers and percentages of EV-A71 neutralizing IgM mAbs, non-neutralizing EV-A71 binding IgM mAbs and non-binding IgM mAbs. (D) EV-A71 binding activity of 56 IgM mAbs were determined by ELISA using mAb containing culture supernatants. EC50 (half maximal effective concentration) values for IgG1 mAbs and half-maximal binding dilution of IgM mAb containing culture supernatants were calculated using GraphPad Prism 7.
Table 1.
EV-A71 neutralizing mAbs.
Fig 2.
Binding activity of representative IgG1 and IgM mAbs with EV-A71.
(A) ELISA was conducted using purified mAbs and mature EV-A71 subgenotype C4a virions; data shown are EV-A71 specific IgG1 and IgM mAbs and a poor binding IgG1 mAb (M2-10). The curves were fitted by nonlinear regression. (B) Heatmap showing ELISA and neutralizing antibody EC50 (half maximal effective concentration) values. (C) Immunoprecipitation of EV-A71 virions with mAbs to measure the binding of IgG1 and IgM antibodies to EV-A71. IgG mAb 16-3-10B was used as a positive control. 50% effective concentration (EC50) values were calculated using GraphPad Prism 7 software.
Fig 3.
Heavy chain variable gene signatures in EV-A71 infected HFMD patients.
(A) Maximum-likelihood phylogenetic tree of heavy chain variable region (VH) for all of the 145 antibodies screened was generated and arranged by IGH variable gene usage. Antibody ID and isotypes shown in bold red are EV-A71 neutralizing antibodies; those in blue are non-neutralizing EV-A71 binding antibodies; those in black are EV-A71 non-binding antibodies. The phylogenetic tree was generated by using MEGA 11. (B) Sequence alignment of heavy chain variable genes of M1-24 and M3-7. Amino acids are colored according to similar properties: blue, acidic; red, basic; green, hydroxyl; brown, amine; yellow, sulfhydryl; gray, aliphatic; orange, aromatic; pink, imino acid. Consensus across mAbs heavy chains is indicated below each amino acid residue by symbols: no symbol, conserved residue; asterisk, non-conserved residue.
Fig 4.
Neutralization of EV-A71 infection at pre-attachment and post-attachment stages by IgG1 and IgM mAbs.
Inhibition of EV-A71 (C4a) infection at pre-attachment (red) and post-attachment (black) stages were determined by neutralization assay; cell viability was determined using Cell Counting Kit-8. (A) mAbs neutralize EV-A71 at post-attachment stage at much higher concentrations than at pre-attachment stage. (B) mAb neutralizes EV-A71 at similar concentrations at two stages. The curves were fit by nonlinear regression. Half-maximal inhibitory concentrations (IC50) were calculated using GraphPad Prism 7 software.
Fig 5.
Epitopes on EV-A71 critical for neutralization.
(A) EV-A71 Subgenotype C4a and C4b escape variants were selected by virus propagation in RD cells in the presence of excess mAbs. Escape variant strains were plaque purified and the P1 region was sequenced. We identified variants with substitutions at six positions on VP1 and VP2 proteins, which escape the neutralizing mAbs. (B) The residues critical for antibody neutralization and the SCARB2 binding site are mapped on EV-A71 virion surface. VP1 protein is colored in black, VP2 colored in gray, and VP3 colored in light gray. Residues critical for neutralization of M1-16 are colored in red; residue critical for M1-17 and M3-7 neutralization colored in blue; residue critical for M1-20 neutralization colored in cyan; residue critical for M1-24 neutralization colored in orange. The picture was created with PyMOL based on EV-A71 structure 3VBS [33]. (C) Cross-neutralization of mAbs, combining mAbs and escape variants of this study and those reported by Huang et al. [17], were determined by using wild-type and escape variant strains of EV-A71 C4a. Antibodies are grouped according to their cross-reactivity with the variants.
Fig 6.
Epitope specific antibody response in EV-A71 infected HFMD patients.
Neutralization titers against wild-type and escape variants of EV-A71 C4a in acute phase sera from 28 EV-A71 infected HFMD patients were tested. Serum samples showing a > 2-fold decrease in titer against variant(s) compared with wild-type were defined as sensitive to that variant(s) and results from these samples are highlighted in orange. Amino acid substitution in each variant and numbers of serum samples sensitive to each variant are shown above. Black lines show neutralization geometric mean titers of each column. The Wilcoxon signed rank test was used to compare log2 NAb titer against wild-type and variant strains for all samples tested (***: p<0.001).