Research ArticleVIROLOGY

Discovery and structural characterization of a therapeutic antibody against coxsackievirus A10

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Science Advances  19 Sep 2018:
Vol. 4, no. 9, eaat7459
DOI: 10.1126/sciadv.aat7459
  • Fig. 1 Stability and immunogenicity of CVA10 particles.

    (A) The SDS-PAGE analysis and negative staining EM of the three types of CVA10 particles. Lane 1, mature virions; lane 2, A-particles; lane 3, procapsids; and lane 4, molecular mass (MM) markers (in kilodaltons). Protein components of each type of particles are labeled along the right side of each lane. The genome is present in both mature virions and A-particles but absent in procapsids. (B) The genome exposure and thermal stability of CVA10 particles determined by PaSTRy. The high fluorescence value throughout the whole temperature range indicates that the genome in the A-particle is more exposed than the one in the mature virion. (C) In vitro neutralizing titer of antisera of CVA10 particles. BALB/c mice were vaccinated intraperitoneally with CVA10 mature virions, HI mature virions, A-particles, HI A-particles, procapsids, and adjuvant (control) at weeks 0 and 3. Mice were bled at weeks 0, 2, 4, 6, and 8 for neutralization assays, and the levels of neutralizing titers of antisera are expressed as means ± SD. Statistical significance is determined by Mann-Whitney test (**P < 0.01). (D) In vivo protective efficacy of antisera of CVA10 particles. The infected mice were treated with antisera and were monitored daily for clinical illness and death. All experimental groups showed 100% survival rates, but 0% for two control groups.

  • Fig. 2 Characterizations of NAb 2G8.

    (A) Neutralization assay of CVA10 NAb 2G8. The IC50 value is 0.2 μg/ml. (B) Binding efficacy of NAb 2G8 evaluated with the indirect ELISA. NAb 2G8 exhibits high binding affinities with the CVA10 particles [median effective concentration (EC50) values from 83.1 to 251.1 ng/ml]. The IC50 and EC50 values were calculated with nonlinear regression fitting curves. The optical density (OD) value was read at A450/620. (C) The competitive ELISA of NAb 2G8. The binding of NAb 2G8 to CVA10 particles was significantly blocked by sera from mice immunized with CVA10 and CVA10-positive human sera. The percentages of blocking are expressed as means ± SD. (D) In vivo preventive and therapeutic efficacy of NAb 2G8. The infected mice were treated with NAb 2G8 12 hours before (blue line) or 24 hours after (red line) infection intraperitoneally with CVA10 and monitored daily after inoculation. Both experimental groups showed 100% survival rates, but 0% for two control groups. (E) Neutralization assay of NAb 2G8 at pre-attachment (blue line) or post-attachment (red line). NAb 2G8 exhibits much higher neutralizing capacity at pre-attachment (IC50 = 2.1 μg/ml) than at post-attachment (IC50 = 22.7 μg/ml). (F) Amount of cell-bound CVA10 viruses detected by RT-PCR. Indicated with the relative CVA10 genome level, the amount of cell-bound viruses gradually decreased as a function of increasing NAb 2G8 concentrations in both cases of pre- and post-attachment. Values are expressed as means ± SD. Experiments were repeated in triplicate. PBS, phosphate-buffered saline.

  • Fig. 3 Atomic structures of the CVA10 mature virion, A-particle, and procapsid.

    (A to C) The isocontoured display of cryo-EM density maps (radially colored) of mature virion (A), A-particle (B), and procapsid (C) viewed along a twofold axis. One icosahedral asymmetric unit and icosahedral axes are labeled with a white triangle and numbers. (D to F) Superpositions (a protomer) of the CVA10 mature virion (red), A-particle (blue), and procapsid (cyan). The differences are mainly located at the VP1 N-termini (D), the VP1 loops (BC, DE, HI and GH) close to fivefold axes (E), and the VP3 BC loop (F). (G) Superpositions (a protomer) of mature virions of CVA10 (red), EV71 (orange), CVA16 (magenta), CVB3 (yellow), poliovirus (gray), and EVD68 (green). (H) Cutaway view of all the pocket factors (represented with sticks) in (G) situated in a “pocket” (light blue) of the CVA10 mature virion. (I) Back view of (H) without the pocket density. The CVA10 mature virion resembles CVA16 and EV71 much more than it does CVB3, poliovirus, and EVD68.

  • Fig. 4 Atomic structure of the CVA10 mature virion complexed with Fab 2G8.

    (A) The isocontoured display of the cryo-EM density map (radially colored) of the immune complex CVA10-M-2G8 viewed along an icosahedral twofold axis. One icosahedral asymmetric unit and icosahedral axes are labeled with a white triangle and numbers. (B) A quarter of the central cross section of (A) exhibiting the densities of capsid (yellow to green) and Fab 2G8 (cyan to blue). The radii are marked by the arcs. (C) Surface representation of a protomer in (A) showing the interaction interface between Fab 2G8 (ribbon diagram) and the capsid. Only the variable domain (light chain, orange; heavy chain, magenta) of 2G8 is displayed. The capsid proteins VP1 to VP3 are colored light blue, light green, and light red, but the surface on the capsid covered by the footprint of the Fab 2G8 is respectively highlighted in their darker colors. (D to G) Expanded views of interaction interface spotlighting the VP1 C terminus (D and E), VP3 AB loop (F), and VP2 EF loop (G). Presumable hydrogen bonds and salt bridges in the interaction interface are marked by red dashed lines.

  • Fig. 5 Structure comparisons between three types of CVA10 particles complexed with Fab 2G8.

    (A to C) Views of the regions surrounding an icosahedral twofold axis of immune complex structures of CVA10-M-2G8 (A), CVA10-A-2G8 (B), and CVA10-P-2G8 (C). Two asymmetric units and icosahedral axes are labeled with two black triangles and numbers in (A). Capsid proteins are displayed in ribbon diagrams, while variable domains of Fab 2G8 are displayed in cryo-EM densities (gray). The atomic model of the variable domain is also shown in (A). (D) The superposition of capsid proteins of three immune complexes demonstrating the consistency of the epitope across three types of CVA10 particles. The atomic models of capsid proteins from CVA10-M-2G8 and variable domains of Fab 2G8 are shown in colored ribbons, but capsid proteins of CVA10-A-2G8 and CVA10-P-2G8 are respectively shown in gray and white. (E) Reported epitopes of enteroviruses mapped onto the CVA10 density map showing only the surface region encircling an icosahedral fivefold axis. All these epitopes are classified into four antigenic sites (sites 1 to 4) surrounding two sides of the canyon. Site 1 represents epitopes in EV71 MA28-7 (yellow), CVA6 1D5 (orange), and poliovirus-A12 (lemon). Site 2 indicates the epitopes in EV71 D5 (blue) and EV71 22A12 (marine). Site 3 belongs to the epitope in EV71 E18 (green). The epitope of 2G8 is uniquely located at site 4.

  • Table 1 Statistics of 3D reconstructions and model refinements.

    RMS, root mean square.

    Mature virion
    (CVA10-M)
    A-particle
    (CVA10-A)
    A-particle*
    (CVA10-A*)
    Procapsid
    (CVA10-P)
    CVA10-M-2G8CVA10-A-2G8CVA1-P-2G8
    Data collection
    MicroscopeTitan KriosTitan KriosTecnai F30Tecnai F30Tecnai F30Tecnai F30Tecnai F30
    Voltage (kV)300300300300300300300
    DetectorK2K2Falcon IIFalcon IIFalcon IIFalcon IIFalcon II
    Pixel size (Å)1.361.361.1281.1281.1281.1281.128
    Electron dose (Å2)60602525252525
    Frames60601717171717
    Defocus range
    (μm)
    0.5–4.70.5–4.70.6–4.30.7–4.40.4–5.90.4–5.90.5–5.7
    Reconstruction
    SoftwareRELION 2.0
    Number of used
    micrographs
    17921792532543174917491809
    Number of used
    particles
    17,09266,306941121,15836,55514,42524,764
    Final resolution
    (Å)
    3.43.43.64.03.94.34.2
    Average B factor
    2)
    −153.0−177.9−206.9−271.7−244.0−253.0−276.7
    Model building
    SoftwareCoot
    Refinement
    SoftwarePhenix
    Model statistics
    Correlation
    coefficient
    (around atoms)
    0.84950.85440.86850.86070.86010.8685
    No. of protein
    atoms
    630450834827808250944994
    Ramachandran
    outliers
    0.00%0.00%0.00%0.00%0.00%0.00%
    Ramachandran
    favored
    93.27%92.15%89.7%87.99%90.72%90.18%
    Rotamer outliers0.00%0.18%0.00%0.00%0.00%0.00%
    Cβ deviations000000
    Clashscore2.732.494.714.842.985.78
    RMS (bonds)0.00780.00520.00890.01230.00770.0089
    RMS (angles)1.21.21.31.31.21.3
    EMRinger score4.74.14.13.33.82.8

    *A-particle from the middle band.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/9/eaat7459/DC1

      Fig. S1. Cryo-EM micrographs of CVA10 particles and CVA10-2G8 complexes, and 3D classification of CVA10 mature virion particles.

      Fig. S2. Structure comparisons of A-particles of CVA10, EV71, CVA16, and CVA6.

      Fig. S3. Global and local resolution analyses of 3D reconstructions of CVA10 particles and CVA10-2G8 complexes.

      Fig. S4. Central sections of density maps of CVA10 particles and CVA10-2G8 complexes.

      Fig. S5. ESPript representation of structure-based sequence alignments of VP1, VP2, VP3, and VP4 of CVA10 with six other representative picornaviruses.

      Fig. S6. Structure comparisons of the canyon regions between CVA10 and other enteroviruses.

      Fig. S7. The cryo-EM density maps of CVA10-2G8 complexes and roadmap of six Fab 2G8 footprints on the CVA10 mature virion capsid.

      Table S1. Regions with unidentifiable residues in atomic models.

      Table S2. Structure comparisons (RMSD) of CVA10 particles and CVA10 to other enteroviruses.

      Table S3. Interaction contacts in the immune complex CVA10-M-2G8.

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Cryo-EM micrographs of CVA10 particles and CVA10-2G8 complexes, and 3D classification of CVA10 mature virion particles.
      • Fig. S2. Structure comparisons of A-particles of CVA10, EV71, CVA16, and CVA6.
      • Fig. S3. Global and local resolution analyses of 3D reconstructions of CVA10 particles and CVA10-2G8 complexes.
      • Fig. S4. Central sections of density maps of CVA10 particles and CVA10-2G8 complexes.
      • Fig. S5. ESPript representation of structure-based sequence alignments of VP1, VP2, VP3, and VP4 of CVA10 with six other representative picornaviruses.
      • Fig. S6. Structure comparisons of the canyon regions between CVA10 and other enteroviruses.
      • Fig. S7. The cryo-EM density maps of CVA10-2G8 complexes and roadmap of six Fab 2G8 footprints on the CVA10 mature virion capsid.
      • Table S1. Regions with unidentifiable residues in atomic models.
      • Table S2. Structure comparisons (RMSD) of CVA10 particles and CVA10 to other enteroviruses.
      • Table S3. Interaction contacts in the immune complex CVA10-M-2G8.

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