Characterization of mAbs against DENV1-4.

<p>(A) ELISA plates were coated with 1:800 dilutions of polyclonal rabbit anti-DENV hyper-immune sera at 4°C for 24 hours. After blocking, DENV1-4 viral supernatants were added, which were then detected by incubation with 1 μg/ml mAbs at RT for 2 hours. Next, the plates were incubated with HRP-conjugated goat anti-mouse IgG, and developed using OPD. The OD at a wavelength of 490 nm was measured. The cutoff values are represented by dotted lines. (B and C) BHK-21 cells were infected with DENV1-4, respectively. After 2 days, the infected cells were detected with mAbs. DD1-4, DD3-7, DD4-3, DD5-1, DD9-4, DD13-4, DD17-4, DD27-8, DD30-4, and DD31-3 are DENV4 serotype-specific mAbs. DD7-8, DD11-4, DD14-1, DD15-2, DD18-5, and DD33-2 are cross-reactive mAbs. 4G2 is the positive control.</p

Examination of the neutralizing activity of mAbs against DENV.

<p>(A) <i>In vitro</i> neutralizing activity of mAbs against DENV1-4 was examined using the plaque reduction neutralization test (PRNT). DENV1-4 was incubated with DD11-4 or DD18-5, and used to infect BHK-21 cells. After 4 days, the cells were fixed and stained, and viral plaques were counted after washing. The inhibition percentages were shown. (B) DENV1-4 were incubated with DD11-4 or DD18-5 at 40 μg/ml, and the mixtures were then used to infect BHK-21 cells. After 4 days, viral plaques were counted and the inhibition percentages were determined. NMIgG was used as a control. Data shown are from one representative experiment of two independent experiments.</p

Determination of the binding regions of mAbs by Western blotting.

<p>Four serotypes of DENV antigen (D1 to D4) from infected C6/36 cell lysates were analyzed and detected with mAbs by Western blotting. (A) The E proteins (about 53 kDa) of DENV4 were recognized by DD1-4, DD4-3, DD5-1, DD9-4, DD13-4, DD17-4, DD27-8, DD30-4, and DD31-3. (B) The E proteins of DENV3 and DENV4 were recognized by DD14-1 and DD15-2. The E proteins of DENV1-4 were recognized by DD11-4 and DD18-5. (C) The dimeric form of the NS1 proteins (about 75 kDa) was recognized by DD3-7, DD7-8, and DD33-2. DB16-1 was used as a positive control for dimeric NS1 proteins. (D) DD1-4, DD11-4, DD13-4, DD14-1, DD18-5, DD30-4, and DD31-3 recognized the EDI-II protein (about 36 kDa) of DENV4. The 4G2 mAb (which targets the EDI-II protein of DENV4) was used as a positive control. NMIgG was used as a negative control. (E) DD17-4 and DD27-8 recognized the EDIII protein (about 17 kDa) of DENV4. NMIgG was used as a negative control.</p

Evaluation of <i>in vitro</i> and <i>in vivo</i> ADE with cross-reactive mAbs.

<p>(A) DENV2 S221 (MOI = 5) was incubated with the serially-diluted mAbs for 1 hour at 4°C and then used to infect K562 cells. After 3 days, the cells were stained with 4G2, and analyzed by flow cytometry. (B) K562 cells were incubated with DENV2 S221 (MOI = 5) and the indicated mAbs at 100 μg/ml, and the percentage of infected K562 cells was determined at 72 hours post-infection by staining with 4G2, followed by flow cytometry. NMIgG was used as a control. Unpaired Student’s <i>t</i> tests were used to calculate <i>P</i> values (**P<0.01). (C) AG129 mice were i.v. infected on day 0 with 1 × 10⁵ pfu DENV2 S221 and i.p. treated on days -1 and 1 with 5 μg of DD11-4 (n = 7), DD18-5 (n = 7), or NMIgG (n = 8). The survival rates were recorded for 30 days. Kaplan-Meier survival curves and <i>P</i> values are shown (***<i>P</i><0.001, compared to NMIgG). Data shown are from one representative experiment of two independent experiments.</p

Identification of the epitope residues on E protein.

<p>BHK-21 cells were transfected with vectors expressing wild-type or mutant DENV4 E proteins. After 2 days, the cells were reacted with DD11-4 (A) or DD18-5 (B), and then analyzed by flow cytometry. The binding index of a mAb to a mutant E protein was measured. The binding indices of DD11-4 and DD18-5 were reduced by mutations at W212 and E26, respectively. Data shown are from one representative experiment of two independent experiments. (C and D) Various wild-type or mutant DENV1-3 E proteins were expressed in BHK-21 cells. After fixation and permeabilization, the cells were incubated with mAbs, and analyzed by flow cytometry. The binding index of a mAb to a mutant E protein was measured. Mutations of W212 (W210 in DENV3) and E26 led to a significant loss of binding activity of DD11-4 (C) and DD18-5 (D), respectively. (E) The epitope of DD11-4 is located at residue W212 in EDII. (F) The epitope of DD18-5 is located at residue E26 in EDI. The model is based on the DENV2 E protein model (PDB: 1OAN). EDI is shown in red, EDII is shown in yellow, and EDIII is shown in blue.</p

Alignment of amino acid residues recognized by DD11-4 and DD18-5 in EDI-II protein^a.

<p>^a The EDI-II protein sequences of strains of DENV1-4 were aligned. Single letter amino acid abbreviations are shown.</p><p>^b The epitope residue of DD18-5 is shown in boldface type.</p><p>^c The epitope residue of DD11-4 is shown in boldface type.</p><p>^d The epitope residue of DD11-4 is W210 in DENV3, and is shown in boldface type.</p><p>Alignment of amino acid residues recognized by DD11-4 and DD18-5 in EDI-II protein<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136328#t002fn001" target="_blank">^a</a>.</p

Original images underlying Fig 1C.

Correction: Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2</p

Original images underlying Fig 1D.

Correction: Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2</p

Characterization of mAbs against DENV.

<p>(A) C6/36 insect cells were infected by DENV-1, -2, -3 and -4 or uninfected (Mock). After fixation and permeabilization, mAbs were incubated with cells and binding was assessed by cellular ELISA. <i>A</i>₄₉₀, optical density at 490 nm. (B) Identification of mAbs by Western blotting. C6/36 cells were infected with DENV-1 to -4 (D1, D2, D3 and D4) as viral antigens. Protein samples were dissolved in native sample buffer and fractionated by 10% SDS-PAGE. mAbs recognized E protein (53 kDa) of DENV. (C and D) mAbs recognized DENV-2 E protein and E-DI-II was determined by IFA, respectively. (E) Dissection of DENV-2 mAbs recognized E-DI-II or E-DIII by Western blot analysis. The DENV-2 recombinant E-DI-II-flag (36 kDa) and E-DIII-flag (17 kDa) fusion proteins were expressed in <i>Escherichia coli</i>. Protein extract was dissolved in denatured sample buffer and fractionated on 12% SDS-PAGE. 4G2, a cross-reactive mAb and 3H5, a DENV-2 serotype-specific mAb recognized D2-E-DI-II and D2-E-DIII, respectively. They were used as positive controls.</p

Construction and characterization of humanized DB32-6 mAb.

<p>(A) Amino acid sequences of humanized DB32-6 (hDB32-6). FR, framework region; CDR, complementarity determining region. Red residues represent the different amino acids from murine DB32-6 (mDB32-6). (B) mDB32-6 and hDB32-6 mAbs recognized DENV-2-infected BHK-21 cells by IFA. Cells were counterstained with DAPI (blue) and observed at 400× magnification. (C) Binding activity of hDB32-6 mAbs. Three stable clones of hDB32-6 (hDB32-6-30, hDB32-6-48 and hDB32-6-51) recognized DENV-2-infected C6/36 cells and recombinant E-DIII of DENV-2 by ELISA. (D) Various concentrations of mDB32-6 and hDB32-6-48 mAbs were reactive to DENV-2 and recombinant E-DIII of DENV-2 but not to mock control. NMIgG and NHIgG were used as negative controls. (E) Binding affinities of mDB32-6 and hDB32-6-48 to E-DIII of DENV-2. mAbs affinity analysis was performed by surface plasmon resonance (SPR). Binding affinity was tested at the mAb concentrations ranging 0 to 4 nM. Binding curves and kinetic parameters are shown.</p