Figure 1.
Characterization of mAb 2A10G6 in vitro.
A. Cross-reactivity of 2A10G6 with four DENV serotypes and four other representative flaviviruses determined by indirect immunofluorescence analysis. BHK21 cells were infected separately with DENV1–4, YFV, WNV, JEV, or TBEV. Three to 5 days after infection, cells were fixed and analyzed by IFA with 2A10G6. Uninfected cells were run simultaneously as negative controls. B. The specificity of 2A10G6 for the DENV2 prME protein. BHK21 cells were transiently transfected with the recombinant plasmid encoding the prME protein or with a control vector, empty pcDNA3.1. At 48 h after transfection, cultured cells were lysed and analyzed by western blotting with 2A10G6. C. The specificity of 2A10G6 for the DENV2 DI–DII peptide. The DENV2 DI–DII peptide was expressed in E. coli as a fusion protein with thioredoxin, and binding of different concentrations of 2A10G6 with DENV2 antigen and recombinant DI–DII peptide was detected by ELISA.
Figure 2.
Comparison of the amino acid sequences of flavivirus E proteins.
Amino acids at positions 72 to 120 are shown. Boxed residues indicate that positions 98 (green), 99 (blue), and 101 (red) were highly conserved among the flaviviruses examined and constitute a conformational epitope.
Figure 3.
Structural mapping of the 2A10G6 epitope.
A. the structure of the 2A10G6 epitope in the crystal structure of DENV2 E protein. Residue numbering is based on the E gene sequences. The regions in red are thought to be structurally near these three residues. B. Three-dimensional comparisons of the 2A10G6 epitope in the three known crystal structures. DENV2, WNV, and TBEV E proteins are in shades of red, blue, and cyan, respectively.
Figure 4.
Neutralization activity of 2A10G6 to various flavivirus strains.
Viruses were mixed with 2A10G6 at different concentrations. Neutralization activities were evaluated by plaque reduction assays using BHK21 cells.
Figure 5.
Protection of 2A10G6 against DENV 1-4 in suckling mice.
2A10G6 at 100, 20, or 4 µg/ml was incubated with 104 PFU/ml of DENV 1–4 and inoculated into brains of suckling mice. Mortality was the outcome measured. Viruses with PBS were included as a negative control. The number of animals for each antibody dose ranged from 9 to 12. Kaplan–Meier survival curves were analyzed by the log-rank test and compared to curves of the PBS controls. Significant differences are indicated by asterisks (*** p<0.001, ** p<0.01 and * p<0.05). A, B, C, and D represent DENV1, DENV2, DENV3, and DENV4 infection, respectively.
Figure 6.
Protetcion of 2A10G6 against WNV in mice.
Group of 4-week-old Balb/C mice were administrated with 200 µg of 2A10G6 one day before (A) or after (B) challenge with 40 PFU of WNV. PBS was included as a negative control. Kaplan–Meier survival curves were analyzed by the log-rank test and compared to curves of the PBS controls. Significant differences are indicated by asterisks (** p<0.01 and * p<0.05).
Figure 7.
Mechanism of 2A10G6-mediated neutralization.
A. Dengue virus binding to BHK21 cells. The DIII-specific neutralizing mAb 2B8 blocked cellular attachment significantly more than the DI–DII–specific neutralizing mAb 2A10G6 or controls, including no antibody (BSA) and the non-neutralizing mAb 2H11. Fold reductions are reported with standard deviations as the average of four independent experiments. B. Pre- and post-adsorption inhibition assays. Pre-binding of DENV2 with 2A10G6 or 2B8 significantly protected against infection (solid lines) (Pre). In contrast, 2A10G6 but not 2B8 inhibited infection when added after virus binding (dotted lines) (Post). Infection percentages at the different antibody concentrations are shown with standard deviations.