Serotype specific epitopes identified by neutralizing antibodies underpin immunogenic differences in Enterovirus B

Fig. 1. Characterizations of the MAbs 6C5 and 4B10. a Dose-dependent binding analysis of MAbs (6C5 and 4B10) against representative enterovirus B members by ELISA. Each plot represents the mean of OD450 values from triplicate wells. Error bars represent mean ± SD. b BIAcore SPR kinetic profile of MAb 6C5 (left) and 4B10 (right) against E30 virus. The binding affinity KD (equilibrium dissociation constant, KD = Kd/Ka, where Kd and Ka represents the dissociation rate constant and association rate constant, respectively) values were obtained by using a series of MAb concentrations and fitted in a global mode in each sensorgram. c Competitive binding between 6C5 and 4B10. In the upper panel, MAb 6C5 was injected first, followed by the second injection of 4B10 or 1A1 specific to E6 as control. In the lower panel, 4B10 was injected first, which was followed by 6C5 or 1A1; the control groups are carried out as the other lines exhibit. d Neutralization of E30 by 6C5 (top) and 4B10 (bottom) using plaque-reduction neutralization test (PRNT). Neut50 values indicate concentration of antibody required to neutralize fifty percent of the viral titer. The Neut50 of 6C5 IgG, 6C5 Fab, 4B10 IgG and 4B10…

Fig. 2. Blocking of viral attachment receptor CD55 and uncoating receptor FcRn by 6C5 and 4B10. a BIAcore SPR kinetics of FcRn or CD55 binding to E30. The RU curves are fitted globally to calculate the KD value for FcRn and CD55. b, c Competitive binding between 6C5 or 4B10 and CD55 (left) or FcRn (right). In the upper two diagrams of each panel, 6C5/4B10 was injected first, followed by either CD55, or FcRn, or ICAM5, the uncoating receptor for EVD68; in the lower two diagrams of the counterparts, CD55 or FcRn was injected first, followed by either 6C5, or 4B10, or 1A1. The related control groups were carried out as the other curves show. d Amount of virions remaining on the cell surface, as detected by real-time PCR, when exposed to 6C5 (left) or 4B10 (middle) or the mixture of 6C5 and 4B10 (right) before or after the virions attach to RD cells and the results here were expressed as mean ± SD.

Fig. 3. Cryo-EM structures of E30 in complex with 6C5 Fab or 4B10 Fab. a Surface representations of E30-6C5-complex (left) and E30-4B10-complex (right). The viral parts of both complexes are rainbow-colored as the color bar below shows; the 6C5 Fab and 4B10 Fab are colored in cyan and yellow, respectively. b Electron density maps for representative sections of VP1 and 6C5 (left) from E30-6C5-complex, and sections of VP2 and 4B10 (right) from E30-4B10-complex. c, d Fab occupancy (left) and epitopes (right) of 6C5 (c) and 4B10 (d) on a viral pentamer. The pentamers are shown as surface (left) or cartoon (right) in the signature colors (VP1, blue; VP2, green; VP3, red), while Fabs are colored in the same scheme as in 3a. The epitopes of 6C5 (left in 3c) and 4B10 (right in 3d) are shown as spheres and those from one protomeric unit are circled by black dotted lines. e Side views of two Fabs bound to a pentamer. The same color scheme is applied as above. When the E30-6C5 (top) and E30-4B10 (middle) are superposed, the complex (bottom) generated shows an angle of 53.8° between 6C5 and 4B10. f Footprints of 6C5 and 4B10 on the sterographic projection of E30. Residues of VP1, VP2 and VP3 are…

Fig. 4. Interactions between E30 and 6C5/4B10. a Platform for Fab (6C5, left and 4B10, right) binding with E30. Loops from E30 involved in interactions and the Fab parts are shown as cartoon, while the remaining parts of E30 are shown as surface. Also, the parts from 6C5 and 4B10 interacting with E30 are colored in dark cyan and dark yellow, respectively. The color scheme is as in Fig. 3c. b Binding interface between E30 and 6C5 (left), as well as E30 and 4B10 (right). Residues involved in the binding are shown as sticks and hydrogen bonds are shown as orange dashed lines. c Sequence conservation analysis. The loops from E30 that are involved in interactions between E30 and 6C5 (VP1 BC loop), as well as E30 and 4B10 (VP1 C-terminus, VP2 EF loop and VP3 C-terminus) are aligned with those of other enterovirus B members (E1, E6, E7, E11, E18, CVB3, CVA9), and colored according to sequence conservation as listed in the table below. d Structure conservation analysis. The same loops in 4c colored in the signature color scheme are superposed with their counterparts from E6 (colored in wheat), CVB3 (colored in pale cyan), and CVA9 (colored in pale magenta).

Fig. 5. Mechanism of neutralization of E30 by NAbs 6C5 and 4B10. a, b Clashes between Fab 6C5 (a)/4B10 (b) and E30 cellular receptors – CD55 (left)/FcRn (right). The viral pentamer, Fabs 6C5 and 4B10, and receptors CD55 and FcRn are colored in grey, cyan, yellow, magenta, and orange, respectively. When E30-receptor-complex is superposed with E30-Fab-complex, clashes between each Fab and each receptor are prominent and marked with star symbols. c Roadmap exhibiting the footprints of FcRn, CD55, 6C5 and 4B10 on the viral surface. The footprints of CD55, 6C5 and 4B10 are colored in magenta, cyan, yellow, respectively in the left map, and the footprints of FcRn, 6C5 and 4B10 are colored in blue, cyan, yellow, respectively, in the right map, where canyons are shaded by light shadows. Overlapped footprints between receptors and 4B10 are colored in green and outlined with white lines, while the ones between receptors and 6C5 are colored in purple and outlined with white lines.

Fig. 6. Classification of the enterovirus-Fab-complexes. All published complexes of enterovirus and neutralizing MAb were utilized for comparison. The complexes can be classified into four patches, a patch 1—around the north rim of the canyon, including E30-6C5, CVA6-1D5 (PDB CODE: 5XS7) and EVD68-11G1 (PDB CODE: 6AJ9), b patch 2—around the south rim of the canyon, which includes EV71-D6 (PDB CODE: 5ZUD), CVA10-2G8 (PDB CODE: 6AD0), EV71-22A12 (PDB CODE: 3J91) and EV71-D5 (PDB CODE: 3JAU), c patch 3—near the threefold axis constituted by EV71-E18 (PDB CODE: 4C0U), EV71-A9 (PDB CODE: 5ZUF), EVD68-15C5 (PDB CODE: 6AJ7) and HRVB14-C5 (PDB CODE: 5W3E), and d patch 4—inside the canyon, including E30-4B10, PV1-A12 (EMDB CODE: 5670) and PV2-A12 (EMDB CODE: 5671).