1. Volume 26, Issue 1, Pages 51-59.e4 (January 2018)
Structural and Functional Characterization of a Cross-Reactive Dengue Virus Neutralizing Antibody that Recognizes a Cryptic Epitope 
Jie Li, Daniel Watterson, Chiung-Wen Chang, Xiao-Yan Che, Xiao-Quan Li, Daniel J. Ericsson, Li-Wen Qiu, Jian-Piao Cai, Jing Chen, Scott R. Fry, Stacey T.M. Cheung, Matthew A. Cooper, Paul R. Young, Bostjan Kobe 
Structure 
Volume 26, Issue 1, Pages e4 (January 2018)
DOI: /j.str
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2. Structure 2018 26, 51-59.e4DOI: (10.1016/j.str.2017.11.017)
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3. Figure 1
Neutralization Curves for mAbs 3E31 and 4G2 for all Four DENV Serotypes
DENV-1–4 are shown in (A–D). Data were derived from a PRNT assay and percentage neutralization was calculated relative to untreated virus. Data are represented as means ± SEM.
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4. Figure 2
Crystal Structure of the Fab Fragment from mAb 3E31 in Complex with DENV-4 DIII Protein
(A) Crystal structure of DENV-4 DIII (blue) in complex with Fab fragment from 3E31. The heavy-chain and light-chain framework regions are colored black and gray, respectively, with the CDR loops color coded and labeled H1–H3 and L1–L3. The variable and constant domains of the heavy and light chains are indicated.
(B) Close-up view of interacting residues. This epitope is recognized by five of the six complementary-determining regions (CDRs; H1, H2, H3, L1, and L3). The 3E31 epitope comprises the AB loop (residues 314–319) and β strand E (residues 365–370), as well as residues 321, 323, and 352.
(C) Gln316 and His317 (AB loop), and Glu368 and Glu370 (β-strand E) form hydrogen bonds with the Fab and are strictly conserved in DENV-1–4. DENV-4 DIII residues, which use their side chains to form salt bridges or hydrogen bonds with Fab 3E31, are marked with yellow asterisks. Residues with 100% conservation between the four DENV serotypes are shown in blue.
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5. Figure 3 3E31 Recognizes a Cryptic DENV Epitope
(A) mAb 3E31 was used to capture DENV-2 virions at various temperatures. Virion binding was detected using the FP-specific mAb 4G2. Increased binding was observed at higher temperatures.
(B and C) Virus neutralization is temperature and time dependent. Virus and antibody were incubated at varying temperatures or times prior to addition to cells. Higher temperature or extended incubation resulted in significantly increased neutralization. Percentage neutralization was calculated relative to untreated virus at the matched temperature. Binding and neutralization data are represented as mean ± SEM.
(D and E) The structure of the E protein dimer within the mature virion structure (D). One monomer within the dimer is colored, with domains I, II, and III shown in red, yellow, and blue, respectively. The hydrophobic FP is colored green. Dashed box designates the inset representations of the binding footprints of 3E31 within the context of the E dimer on the mature virion shown in (E), where the color scheme of domains is as in (D), with the 3E31 epitope footprint shown in cyan. One E monomer within the dimer is shown in surface representation, with domains I and II transparent to reveal the hidden epitope on DIII.
(F) Epitope locations on virus particles at different stages of the virus life cycle. Surface representations of immature virion (pr peptide removed for clarity); low-temperature mature virion; virion in Fab 1A1D-2-bound “breathing” state (Fab fragments omitted); and the open, high-temperature (37°C) mature virus. Individual subunits are shown in shades of gray with epitope footprint in cyan as per (E). The asymmetric unit of each virion structure is shown in inset below for clarity. The 3E31 epitope is completely obscured in the mature and open virion forms, and mostly buried in the immature and breathing forms. Virion structures are based on PDB: 4B03 (Kostyuchenko et al., 2013) superimposed with PDB: 3C6E (Li et al., 2008), PDB: 3J27 (Zhang et al., 2013a), PDB: 2R6P (Lok et al., 2008) superimposed with PDB: 1OAN (Modis et al., 2003) (DIII fitted separately to EDI and EDII), and PDB: 3J35 (Zhang et al., 2013b) fitted with PDB: 3J27, respectively.
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6. Figure 4 3E31 Blocks DENV E-Mediated Cell Fusion
(A) DENV-2-infected C6/36 cells were exposed to mAbs during acidification to pH 6.0, which triggers syncytia formation in DENV-infected cells (outlined and labelled with star for clarity in the virus control panel). Live cells were visualized using time-lapse bright-field microscopy. Both 3E31 and 4G2 inhibited fusion at 0.5 mg/mL.
(B) Quantification of fusion using a cell counting algorithm shows reduction of fusion for 4G2, 3E31, and 3E31 Fab but not non-specific antibody control (all 0.5 mg/mL).
(C) 3E31 fusion inhibition is dose dependent. Varying concentrations of 3E31 were added together with low-pH media and fusion assessed as per (B). Data are represented as mean ± SEM.
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7. Figure 5 Antibody-Dependent Enhancement Assays
ADE activity of (A) mAb 3E31 and (B) mAb 4G2 (positive control) tested on K562 cells. Only minor enhancement was observed for 3E31 in Fc-receptor-expressing cells. By contrast, binding by 4G2 led to ADE in all four DENV serotypes in a dose-dependent manner. Data are represented as mean ± SEM.
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