Structural basis for hepatitis E virus neutralization by potent human antibodies

Fig. 1.. Human HEV memory B cell antibodies. (A) ELISA graphs showing the reactivity of purified serum IgGs (blue) and IgAs (red) from HEV-exposed donors against HEV-CA protein (left). The heatmap comparing the area under the curve (AUC) values derived from the ELISA titration curves (right). The black line corresponds to the negative control (Ctr-, seronegative donor). (B) Graph comparing the in vitro neutralization activity of purified IgGs against naked HEV virions (HEV-3c) (left). Calculated IC50 values are presented on the heatmap (right). (C) Heatmap comparing the AUC values for the ELISA binding of purified IgGs to HEV-CA domain proteins. (D) Flow cytometric plots showing the percentage of circulating blood HEV-CA+ IgG memory B cells in HEV-exposed individuals. (E) ELISA graphs showing the reactivity of the HEV memory B cell antibodies against HEV-CA protein (left). The pie charts (middle) show the proportion of antibodies binding to the HEV-CA protein. The number of tested antibodies is indicated in the center of the pie chart. EC50 values calculated from the titration curves are represented in the dot plots (right). (F) Pie charts showing the distribution of clonally expanded…

Fig. 2.. Reactivity profiles of human HEV antibodies and their corresponding immunoglobulin gene repertoire. (A) Heatmap showing the reactivity of anti-HEV-CA IgG and IgA memory B cell antibodies (n = 144) against HEV g3-2712 capsid domains, Paslahepevirus HEV-CA (genotypes 1d to 8) and Rocahepevirus CA (HEV-C1) proteins, with color-coded cells according to the values shown in table S2. Black dots on the right indicate IgA antibodies. (B) Bar graph and pie chart showing the proportion of IgG antibodies (n = 138) recognizing the P domain for each HEV-exposed donor (left) and in total (right), respectively. (C) Pie charts comparing the distribution of VH and JH gene usage of anti-CA P domain (CAP), anti-CA MS domains (CAMS) and control (IgG.mB) IgG memory B cell antibodies. (D) Pie charts comparing the κ- versus λ-Ig chain usage of CAP and CAMS IgG antibodies. Groups in [(C) and (D)] were compared using 2 × 5 and 2 × 2 Fisher’s exact test, respectively. (E) Dot plots comparing plots comparing the number VH, Vκ, and Vλ gene mutations of CAP, CAMS, and control (IgG.mB, white) IgG antibodies. (F) Bar graph showing the proportion of IgGs recognizing strictly conformational HEV-CA epitopes on as…

Fig. 3.. Neutralizing activity of human HEV antibodies. (A) Dot plots comparing the percentage of HEV neutralization of human anti-CA antibodies tested in triplicate at 50 μg/ml according to the targeted region (left) and donor (right). Blue-colored dots correspond to P-specific antibodies. (B) Dot plots (left) showing IC50 values of HEV neutralization for anti-P antibodies segregated according to the donors. Pie chart (right) showing the distribution of anti-P antibodies by IC50 values color coded in shades of blue, with darker shades indicating higher potency. (C) Dot plots (top) comparing the number of somatic mutations in VH and VL genes between neutralizing and non-neutralizing anti-P antibodies. Antibodies with IC50 > 50 μg/ml were considered as non-neutralizing. Groups were compared with the two-tailed Mann-Whitney test. Correlation plot (bottom) showing the frequency of somatic mutations in VH and VL genes [f(VH+VL mut.)] versus IC50 values of anti-P antibodies. White dots correspond to non-neutralizing antibodies. The two-sided Spearman rho correlation coefficient and corresponding P value are shown. (D) Competition ELISA heatmap showing the level of binding inhibition to HEV-CA…

Fig. 4.. Structure of HEV capsid P domain complexes with Es1.327, Es4.431, and Es5.127. (A) Surface representation of cryo-EM models of the HEV-CA P domain in complex with Es1.327, Es4.431, and Es5.127 Fabs. Protomers of the P dimer are colored in shades of green. VH and VL chains of each Fab are highlighted with distinctive colors. The black lines indicate the approach angle. White arrows on top views of the complexes indicate the rotation angle of Es4.431 and Es5.127 Fabs relative to Es1.327 Fab. (B) Close-up in ribbon representation of the epitope and paratopes in the P domain–Fab interaction. Hydrogen bonds between chains are represented with dashed lines, and the residues participating in the interactions are indicated. (C) Amino acid sequence alignment of Es1.327, Es4.431, and Es5.127 VH and corresponding germline genes. Residues participating in hydrogen bonds indicated in (B) are highlighted with red boxes. (D) Binding of Es1.327, Es4.431, and Es5.127 Fabs to HEV viral-like particle (VLP). Surface representation of the crystal structure of Paslahepevirus HEV VLP (PDB ID: 3HAG) colored by domains according to (A). Fivefold (pentagon), threefold (triangle), and twofold (oval)…

Fig. 5.. Molecular determinants of cross-genotype binding by group VI anti-P HEV antibodies. (A) SPR sensorgrams (left) comparing the apparent affinity of purified neutralizing anti-P IgG antibodies (n = 5) for the binding to genotype 1d (purple) and 3a (blue) HEV-CA proteins. Corresponding calculated KD values are indicated in the dot plot on the right. RU, response units; FD, fold difference between KD values obtained with genotype 1 and 3 HEV-CA proteins. (B) Modeling of amino acid variations in the RS loop of the P domain across different HEV genotypes. Ribbon representations of AlphaFold-predicted P domain structures (green) aligned to the cryo-EM model of the HEV P domain genotype 3 in complex with Es1.327 (blue) or Es4.431 Fabs (brown). Dashed lines indicate hydrogen bonds identified in the cryo-EM structures. HEV-CA UniProt accession numbers: P29326, Q03500, Q9YLQ9, A0A348BSN0, BCD83331.1, BFL88677.1, UEC95198.1, and MH410174. Amino acid variants are shown as purple stick representations and highlighted with purple boxes in the sequence alignment (bottom left). (C) Heatmap comparing the ELISA binding (as AUC values) of group VI anti-P antibodies to purified recombinant genotype 3…

Fig. 6.. Cryo-EM reconstruction of HEV-C1 P domains in complex with Es5.127. (A) Comparison of Paslahepevirus (cryo-EM) and Rocahepevirus (AlphaFold) HEV P domains. Left and middle panels: Surface representation of P colored by electrostatic potential on a scale from −5 (negative charge) to 5 (positive charge) kT/e. Black line delimits the interaction zone of Es5.127 Fab with Paslahepevirus P domain. Right panel: Close-up views of RS and XY loops in ribbon representation of Paslahepevirus and Rocahepevirus P domain. Side chains of these loops are showed in stick representation. (B) Fitting of the model of Paslahepevirus P/Es5.127 complex into the cryo-EM reconstruction of Rocahepevirus HEV P/Es5.127 complex. (C) SPR sensorgram showing the apparent affinity of the anti-HEV P domain IgG antibody Es5.127 for binding to purified HEV-C1 protein. The corresponding calculated KD value is indicated in the graph (top left).