Fig 1.
Cryo-EM maps of different EV71 particles or VLP in complex with D5 Fab or intact IgG.
(A) F-particle in complex with Fab. One icosahedral asymmetric unit of the capsid is indicated by a black triangle. (B) F-particle in complex with IgG. The color bar labels the corresponding radius from the center of the sphere (unit in Å). (C) E-particle in complex with Fab. (D) VLP in complex with IgG. The Fab components of the complexes are rendered in green to blue color. In the IgG bound complexes (F-particle-IgG and VLP-IgG), the density of the Fc region of the antibody could not be resolved owning to its extremely dynamic nature. (E) F-particle-Fab map with fitted models of six adjacent protomers around the 2-fold axis. The Fab densities were removed. VP1, VP2 (VP0) and VP3 structures and densities are shown in blue, green and red, respectively. Same color schema was followed throughout. Positions of the 2-fold, 3-fold and 5-fold icosahedral symmetry axes are indicated as grey oval, triangles, and pentagons, respectively. (F) The segmented density of the VP1 compact region from the F-particle-Fab map with the fitted model. (G) Expanded view of a representative portion of the map and model displayed in Fig 1F.
Fig 2.
Close-up views of the antibody-virus junction.
(A) The side and (B) top views of the cryo-EM density of the junction between the D5 Fab (deep sky blue) and the six adjacent EV71 protomers around the 2-fold axis. Similar color schema as in Fig 1E was adopted for the capsid densities but more transparent, and the Fab density was shown in deep sky blue.
Fig 3.
D5 Fab footprints on the surface of the EV71 particles.
(A-B) Overall and expanded views of the D5 Fab footprints on the F-particle. The surface of the EV71 F-particle is shown as a stereographic projection, in which the polar angles θ and ɸ represent latitude and longitude, respectively. The D5 Fab footprints are indicated by red contour lines. The border of one VP2 (VP0)/VP3/VP1 protomer is outlined by black line. The locations of the 2-fold, 3-fold and 5-fold icosahedral symmetry axes are indicated as black ovals, triangles, and pentagons, respectively. In the expanded view, the amino acid residues of EV71 are denoted. The VP1, VP2 (VP0) and VP3 surfaces are shown in light blue, light green and pink, respectively. (C-D) Overall and expanded views of the D5 Fab footprints on the E-particle.
Fig 4.
Binding interface between the D5 antibody and EV71 F-particle in the F-particle-Fab map.
(A) Overall view of the D5-EV71 binding interface located between the D5 Fab variable region and the VP1 of EV71. Models of heavy and light chains of the Fab, and VP1 of EV71 are shown in light purple, cyan, and blue, respectively. Cryo-EM density is shown in grey. A solid density connecting the EV71 VP1 and the Fab heavy chain is highlighted by dotted black circle. The visualization location with respect to the F-particle-Fab map is illustrated using a small panel in the lower left corner. (B) An expanded view of the D5-EV71 binding interface. Black arrows point to the CDR3 region of D5 heavy chain and VP1 GH-loop of EV71, respectively.
Fig 5.
Effect of mutation of heavy chain CDR3 on D5 scFv binding to EV71 particles and SP70 peptide.
The binding of D5 scFv variants with point mutations at heavy chain CDR3 region to (A) F-particle, (B) E-particle, (C) VLP, and (D) SP70 peptide were determined by ELISA assay. The irrelevant hepatitis B core protein (HBc) was used as negative control. Mean values and standard deviations of duplicate samples are shown.
Fig 6.
Mapping of the D5 epitope to VP1 region and identification of critical residue for D5 binding.
(A) A yeast library expressing a combinatorial library of the entire polyprotein of EV71 on the surface was screened for D5 binding by fluorescence activating cell sorter (FACS) analysis. D5-positive yeast clones were isolated, and 34 of them were sequenced for the insertion. This panel shows the representative results of two rounds of sorting. (B) Alignment of the sequences obtained from the D5-positive yeast clones. The consensus region (HKQEKDLEYG) encoded by the VP1 gene is highlighted in red. (C) D5-resistant mutants were generated by passage of EV71/G082 in the presence of D5 antibody (1 mg/ml) and subsequent plaque purification. Five independent D5-resistant clones were recovered. This panel shows the susceptibility of wild-type and D5-resistant viruses to neutralization by D5 or anti-EV71 VLP mouse sera. (D) Binding activity of wild-type and D5-resistant viruses to D5 or anti-EV71 VLP mouse sera determined by ELISA. An irrelevant protein, BSA, serves as the negative control in the assay. Mean values and standard deviations of triplicate wells are shown. (E) Fitness of the D5-resistant mutants. Same amount (viral RNA genome copy number) of mutant or wild-type viruses was added to RD cells and incubated at 37°C for periods of time as indicated. The data are relative values of viral RNA copy normalized with GAPDH mRNA copy for each treatment. Means ± SD of triplicate wells were shown. Statistical significance was analyzed by two-way ANOVA using GraphPad Prism version 4. (F) Binding ability of D5-resistant mutants to SCARB2. Same amount of mutant or wild-type viruses was incubated with SCARB2-Fc and anti-human Fc IgG conjugated beads at 4°C as described in Methods. The pulled-down viruses were quantified by qRT-PCR. Y axis indicates the percentage of the viral RNA copy number of D5-resistant mutants to that of the wild-type virus. The data are Means ± SD of triplicate wells. Statistical significance was analyzed by the Student’s t-test using GraphPad Prism version 4.
Table 1.
Neutralization activity of D5 on a panel of enteroviruses.
Fig 7.
In vivo protective efficacy of antibody D5.
Groups of mice were administered i.p. with PBS or the indicated mAbs, and one day later, inoculated i.p. with 5 × 105 TCID50 of EV71/MAV-W. The mice were monitored daily for (A) survival and (B) clinical signs for a period of 14 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; 4, death. The data shown are survival rates and mean clinical scores for each group at the indicated time points. The logrank test was used to compare the survival rate between each mAb group and the PBS control group. Statistical significance is indicated as follows: n.s., P ≥ 0.05; *, P<0.05; **, P<0.01.