Fig 1.
Characterization of mAb 1G6 in vitro.
(A) Binding of mAb 1G6 with DENV1-4 by IFA. DENV antigen was detected on day 3 after infection with DENV1-4 with mAb 1G6 and an FITC-conjugated secondary antibody. Cells were counterstained with Evan’s blue, resulting in red fluorescence of cells. (B) Binding of mAb 1G6 with purified DENV1-4 ED3 proteins by western blotting. The cross-reactive mAb 6E1 was used as a positive control. (C) Binding graph of mAb 1G6 with purified DENV1-4 ED3 proteins by ELISA.
Fig 2.
MAb 1G6 mediated neutralization in vitro through blocking viral adsorption.
MAbs with concentrations of 500, 100, 20, 4, 0.8μg/ml were mixed with constant 50 PFU viruses. The results were expressed as percentage of neutralization (% Neutralization) shown with standard deviations. (A) Neutralizing ability of mAb 1G6 against DENV4. The non-neutralizing mAb 6E1 was used as a negative control. (B) Pre- and post-adsorption inhibition assays. Pre-binding of DENV4 with 1G6 significantly protected against infection (P<0.001) (Pre).
Fig 3.
Prophylactic and therapeutic efficacy of 1G6 in suckling mice.
(A) Mice were administered saline or a single 50 μg dose of 1G6 via an IP route one day prior to infection and then infected intracerebrally with 105 PFU/ml of the mouse-adapted DENV–4 virus and monitored for survival for 21 days after infection. (B) Therapeutic efficacy of 1G6 was tested by administering a single 50 μg dose of 1G6 intraperitoneally 4h or 24h after infection with 105 PFU/ml intracerebrally. The number of suckling mice for each group ranged from 10 to 11. Kaplan—Meier survival curves were analyzed by the log-rank test and significant differences are indicated by asterisks (*** p<0.001; * p<0.05).
Fig 4.
(A) Unique mAb 1G6-specific phages after three rounds of bio-panning. The binding of isolated phage clones to 1G6 was examined by ELISA. Data represent the mean ± S.D. of duplicate measurements. (B) Peptide insert sequences (single letter code) enriched after three rounds of selection. Selected peptide sequences are aligned for the consensus motif which is highlighted in bold letters. Binding of 1G6 to control phage clone and binding of control mAb 6E1 with 1G6-specific phage particles were included as specificity controls. Absolute frequency means the times that a specific phage-displayed peptide was identified; while relative frequency means the proportion of a specific phage-displayed peptide normalized by the total number of phage clones. (C) Schematic of generating truncated rED3 proteins. (D) Western blot of purified wild type and DENV4 rED3 truncations. The polyclonal antibody anti-GST was used as a positive control.
Table 1.
Amino acid sequences of DENV-4 ED3 mutants and relative dissociation constants compared to wild-type (Wt) rED3.
Fig 5.
Western blot of 1G6 binding to rED3 with different mutations.
Simultaneous mutations of T388 and H390 abrogated the binding of 1G6 to rED3.
Fig 6.
Amino acid sequence alignment of domain III from DENV1-4 and location of the mAb 1G6 epitope.
(A) Comparison of amino acid sequences of ED3 proteins of wild-type DENV4 and other DENV serotypes. Identical amino acids were indicated by dots, and the unique residues were marker by arrows. (B) Overlay of crystal structures of DENV1-4 ED3. The localization of amino acid substitutions at positions 387–390 in the 3-D structure of monomeric DENV4 ED3, is shown from the side.
Fig 7.
The epitope of mAb 1G6 is highly conserved within and between different genotypes.
(A) Comparison of amino acid sequences of representative ED3 of four DENV4 genotypes. Identical amino acids were indicated by dots. (B-C) Binding of 1G6 with different DENV4 strains within genotype I (B) and representative recombinant ED3 between genotypes (C). MAb binding was detected by ELISA. (D) Neutralization assay of 1G6 against DENV B5 and H241 strains.
Fig 8.
Relationship of 1G6 epitope with the already mapped complex and sub-complex neutralizing epitopes.
The rings represent the area mapped to be the footprint of each mAb on the surface of ED3. The 4E11 epitope is shown in red and includes residues 307–312, 387, 389 and 391. The 9D12 epitope is shown in black and includes residues G304, K305, K307, K310 and P384. The epitope recognized by mAbs 9F12 is shown in purple and includes residues 305, 307, 310 and 330. The epitope for mAb 1G6 is shown in green and includes residues T388 and H390. The diagram is based on the crystal structure of the DENV4 ED3 protein (PDB ID 2H0P).