Article Figures & Data
Figures
- FIG 1
Rs-DIIIs demonstrate favorable binding to DENV MAbs. (A) Domain organization of prefusion homodimeric E glycoprotein. One subunit is color coded by domain (EDI, red; EDII, yellow; and EDIII, blue); the other subunit is shown in gray. DENV-2 is shown as an example from Protein Data Bank (PDB) ID 1OAN (19). (B) Overlay of DENV-1 to -4 EDIIIs bound to the single-chain variable fragment (scFv) of 4E11 from PDB ID 3UZQ, 3UZV, 3UZE, and 3UYP (27). (C) The red spheres indicate library positions on DENV-2 EDIII that were allowed to vary in phage libraries (PDB ID 3UZV [27]). The inset shows the orientation of EDIII, with the A and G strands highlighted. (D) Sequence of rsDIII at the randomized positions. Red indicates substitutions with alanine or serine, and orange indicates other possible substitutions. (E) Reactivity profiles of phage expressing WT DENV-2 EDIII and rsDIIIs toward a panel of DENV MAbs: 2H12 and WNV-E111 (AB loop), 3H5 (FG loop), and M2 (FLAG). Wells coated with BSA served as a negative control. The data are from two experiments performed in duplicate and are plotted as means and standard deviations (SD).
- FIG 2
Biochemical characterization of rsDIIIs indicating abolishment of unfavorable epitopes. (A) Size exclusion chromatogram of WT and rsDIIIs indicating two populations of EDIII, aggregated and monomeric. The monomeric fraction was used in further experiments. AU, arbitrary units. (B) RsDIIIs bind only to MAb 4E11 with high affinity and show little or no binding to MAbs 2H12 and 3H5 by ELISA. In contrast, WT DENV-2 EDIII binds to all three antibodies. The data are graphed as means ± SD from the results of two experiments performed in triplicate. Abs., absorbance. (C) RsDIIIs bind only to MAb 4E11 and demonstrate no binding to MAbs 2H12 and 3H5 by BLI. RsDIIIs were captured on Ni-NTA sensors, followed by antibody association and dissociation. Antibody concentrations ranged from 27 to 0.01 nM for 4E11 and 2H12 and from 6 to 0.01 μM for 3H5. The data were fit to a global 1:1 binding model.
- FIG 3
The core structure of rsDIIIs relative to WT DENV-2 EDIII is maintained. (A) 1H-15N HSQC cross peaks for WT DENV-2 EDIII (black), rsDIII-Ala11 (red), and rsDIII-Ala30 (blue) overlap substantially. Residue labels for WT DENV-2 EDIII peaks that had nearby peaks in the rsDIII-Ala11 or rsDIII-Ala30 spectra are shown. Many cross peaks overlap between the spectra, indicating that the overall fold is similar between WT and rsDIII proteins. (B) RsDIII-Ala11 (red) and rsDIII-Ala30 (blue) residues that overlap assigned WT DENV-2 EDIII resonances were mapped onto the DENV-2 EDIII crystal structure (PDB ID 3UZV [27]). The green spheres represent points of mutation in rsDIIIs relative to the WT.
- FIG 4
RsDIIIs elicit broadly reactive serum responses across DENV serotypes 1 to 4. (A) Immunization and bleed schedule. (B) (Top) Serum endpoint titers against each immunogen reached a maximum by day 28 and remained constant through day 90. (Bottom) Serum antibody responses were not directed toward a hexahistidine tag-bearing control protein. Groups of 10 BALB/c mice were immunized, and serum reactivity was determined for each mouse and plotted as the mean ± SD. (C) ELISA against EDIIIs, with five representative serum samples from each immunogen group. The data are from a single experiment completed in duplicate.
- FIG 5
Engineered EDIIIs elicit potent cross-neutralizing antibodies. (A) Aggregate serum neutralization data for 10 mice against DENV-1 to -4. The lowest serum dilution tested for each sample was 1:20, followed by serial 2-fold dilutions. Naive mice served as the negative control. For DENV-1 to -3, sera from WT DENV-2 EDIII- and rsDIII-Ala30-immunized mice showed similar levels of neutralization, with the IC50s indicated as serum titers. Sera from rsDIII-Ala11-immunized mice demonstrated weak neutralization. Naive sera resulted in partial neutralization at the lowest dilution tested. The data are from two experiments performed in duplicate and are plotted as means ± SD. (B) Neutralization curves for individual mice against DENV-1 to -3 were plotted, and both 50% and 80% focus reduction neutralization titers were determined and plotted. The mean FRNT50 and FRNT80 values for WT DENV-2 EDIII-immunized and rsDIII-Ala30-immunized mice were not significantly different (ns), whereas the mean FRNT50 and FRNT80 values for WT DENV-2 EDIII- and rsDIII-Ala11-immunized mice were significantly different by Kruskal-Wallis one-way ANOVA with Dunn's multiple-comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). For serum samples that did not reach 50% or 80% neutralization at the highest concentration of serum tested (1:20 dilution), a serum dilution of 1:5 was arbitrarily assigned.
- FIG 6
AG129 passive-transfer model of DENV-2 D2S20 infection, and ADE assay. (A) Pooled sera from days 60 and 90 from WT DENV-2 EDIII- and rsDIII-Ala30-immunized mice were transferred to groups of 5 AG129 mice prior to infection with the mouse-adapted strain D2S20 of DENV-2. Pooled naive sera served as the negative control. The mice were monitored until death. All the mice from the WT- and rsDIII-Ala30-immunized groups died on day 5, while naive mice had a mean survival of 7 days. By log rank test, there was a statistically significant trend (P = 0.006) toward shortened survival in the EDIII immunogen-treated groups relative to mice receiving naive serum, suggesting that the immunogen-elicited serum antibodies were insufficient for protection and instead resulted in ADE. The data are from a single experiment. (B) DENV-2 16681 RVPs were incubated with serial dilutions of the indicated mouse sera for 1 h at 37°C, followed by infection of K562 cells. Infection was measured by flow cytometry 48 h later. The fold enhancement was calculated in reference to the minimal infection of K562 cells in the absence of antibody. Shown is one representative experiment of two; the error bars indicate the ranges of duplicate technical replicates.
Tables
- TABLE 1
Apparent dissociation constants for rsDIIIs
Antibody Parameter Value WT DENV-2 EDIII rsDIII-Ala11 rsDIII-Ala30 4E11 kd/ka (nM) 0.01 ± 0.002 0.45 ± 0.11 0.03 ± 0.001 ka (M−1 s−1) (1.4 ± 0.008) × 106 (27 ± 1.2) × 104 (220 ± 1.6) × 104 kd (s−1) (19 ± 2.2) × 10−6 (120 ± 3.9) × 10−6 (54 ± 2.6) × 10−6 2H12 kd/ka (nM) 1.1 ± 19 ka (M−1 s−1) (1.8 ± 3.3) × 106 kd (s−1) (92 ± 2.1) × 10−5 3H5 kd/ka (nM) 120 ± 0.32 ka (M−1 s−1) (1.4 ± 1.4) × 103 kd (s−1) (1.6 ± 0.04) × 10−4
Supplemental material
- Supplemental file 1 -
Table S1 (List of chemical shifts for WT DENV-2 EDIII.)
XLSX, 65K
- Supplemental file 1 -
