Generation and characterization of cross neutralizing human monoclonal antibody against 4 serotypes of dengue virus without enhancing activity

Plasmid construction

Variable HC and LC sequences of B3B9 HuMAb were previously isolated from hybridoma cells and used for construction of HC and LC expression plasmids producing B3B9 rIgG (Pitaksajjakul et al., 2014). This HC plasmid was used as a template for N297Q mutagenesis by site-directed mutagenesis with the In-Fusion Cloning System (In-Fusion^® HD Cloning Plus; Clontech Laboratories Inc., Shiga, Japan). Primers were designed according to the manufacturer’s instructions to mutate the amino acid at position 297 from asparagine to glutamine. This system combines the action of the In-Fusion HD enzyme with inverse polymerase chain reaction (PCR), which generates linearized DNA from a plasmid template. The PCR reaction was composed of the CloneAmp Hifi PCR premix, 300 nM each of forward and reverse primer, 5 ng of plasmid, and distilled water to final volume of 25 µl. The amplification was performed with 35 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 5 s. The inverse PCR products were gel-purified using a PureLink^® Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The linearized plasmids obtained from inverse PCR were then fused by the In-Fusion enzyme, by following the manufacturer’s instructions. The reaction was performed at 50 °C for 15 min. The in-fusion reaction was chemically transformed into Stella chemical competent cells. Individual clones were randomly selected for DNA sequencing.

DNA sequencing and plasmid preparation

The DNA sequencing of the mutated HC plasmids were confirmed by Sanger sequencing (Macrogen Inc., Seoul, Korea). Potential N-glycosylation site of wild type and mutated B3B9 HuMAb was identified using NetNGlyc 1.0 software (Gupta, Jung & Brunak, 2004). Plasmids that contained the target mutation were amplified in E. coli and isolated using a Purelink™ plasmid Midiprep kit (Invitrogen) from 100 ml culture for further transfection in mammalian cells.

Transient expression of N297Q-B3B9 rIgG in HEK293T cells

Plasmids expressing N297Q-B3B9 HC and LC were transfected into HEK293T cells to produce whole rIgG with the N297Q mutation as previously described (Pitaksajjakul et al., 2014). The culture medium containing secreted N297Q-B3B9 rIgG was collected and used in immunofluorescence assays (IFAs) to determine the DENV-binding activity. The N297Q-B3B9 rIgG was purified using a protein A affinity column and measured the concentration of N297Q-B3B9 rIgG by BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). The purity of purified antibody was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

IFA for DENV-binding activity

IFAs were used to determine the binding and specificity of N297Q-B3B9 rIgG for all four DENV serotypes. IFA plates were prepared by infecting Vero cells with DENV at a multiplicity of infection of 0.1 as well as mocked infected. The plates were incubated for 3 days before being fixed and permeabilized with 3.7% formaldehyde and 0.1% Triton X-100, respectively. Culture fluid from transfected cells was added, and the plates were incubated at 37 °C for 1 h. AlexaFluor 488-conjugated anti-human IgG (1:1,000 dilution) (Invitrogen) was added as secondary antibody. The result was observed under fluorescence microscope (IX71, Olympus). For this assay, non-infected cells were used as a negative control.

HuMAb isotyping

The IgG isotype of the N297Q-B3B9 rIgG was determined by PCR as previously described (Omokoko et al., 2014) using complementary DNA isolated from HuMAb clone B3B9 hybridoma as a template. The gene-specific primers used to amplify IgG1, -2, -3, and -4 are listed in Table S1. IgG1 and IgG3 were amplified using the same forward and reverse primers. The primers were used to amplify the hinge region between the Fab and Fc parts that produce different sized PCR products. IgG1 and IgG3 showed specific band at 211 and 346 bp, respectively. With separate PCR reactions, IgG2 and IgG4 were amplified by the same reverse and different forward primers. These IgG2 and IgG4 PCR reactions showed 207 bp and 210 bp, respectively. The PCR reaction was composed of 0.5 µg of cDNA, 1 mM Tris–HCl (pH 8.0), 5 mM KCl, 1.25 mM deoxyribonucleotide triphosphate (dNTP), 1.25 units of ExTaq DNA polymerase (TAKARA, Shiga, Japan), and 5 mM of each primer, with distilled water to a final volume of 25 µl. The amplification of each primer pairs was performed in separate reaction with 35 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 30 s. The PCR products were separated by agarose gel electrophoresis and visualized by staining with SYBR Safe DNA Gel stain (Invitrogen).

Foci reduction neutralization test (FRNT)

FRNT assay was performed as previously described (Pitaksajjakul et al., 2014). DENV at MOI 0.01 were mixed with different concentrations of purified HuMAbs (B3B9 or N297Q-B3B9 rIgG) and incubated at 37 °C for 1 h. Each mixture was added to Vero cells in 96-well cell culture plates and incubated for 2 h. The plates were then overlaid with 2% carboxymethyl cellulose (CMC) in MEM medium with 2% FBS and incubated for 2 days for DENV4 and for 3 days for DENV1, 2, and 3. After that, the cells were fixed with 3.7% paraformaldehyde/PBS and 0.1% Triton X-100/PBS. Immunostaining was performed by an incubation with anti-DENV human antibody, followed by Alexa-conjugated anti-human IgG (H + L) (1:1,000 dilution). Foci numbers were counted under a fluorescence microscope. The percent reduction was calculated by comparing the foci number for each antibody concentration with the number of foci obtained from a virus–PBS mixture (negative control).

Antibody dependent enhancement (ADE) assay on K562 cells

The ADE activity of the N297Q-B3B9 rIgG was also assessed on FcγRIIa-bearing K562 cells (Konishi, Tabuchi & Yamanaka, 2010). Antibodies at serial four-fold dilutions and viruses were mixed in 10% FBS RPMI medium in 96-well poly-L-lysine-coated plates (Corning Inc., New York, NY, USA) and incubated at 37 °C. After 2 h, 50 µl of 2 × 10⁶ cells/ml K562 cells were added. The cell–HuMAb–virus mixture was co-cultured at 37 °C under 5% CO₂ for 2 days. The cells were fixed with an acetone/methanol fixing solution at −20 °C. Immunostaining was performed by adding anti-DENV human antibody and incubating at 4 °C overnight. Horseradish peroxidase-conjugated anti-human IgG ( H + L) diluted in 0.05% Tween and 1% FBS in PBS was then added, and the samples were incubated at 37 °C for 1 h. The signal was developed with a DAB substrate solution (KPL, Gathersburg, MD, USA). Infected cell counts obtained from the test were adjusted with the mean infected cell counts obtained with the four negative controls set in the same experiment. The cut-off value for differentiating enhancing from non-enhancing activities was calculated from the average plus three times the SD of the percentages of infected cells obtained with the four negative controls.

Antibody dependent enhancement (ADE) assay on THP-1 cells

DENV at a multiplicity of infection of 0.1 was incubated with serially diluted antibodies in serum-free RPMI medium at 37 °C for 1 h. Then, 150 µl of 5 × 10⁵ cells/ml THP-1 cells, which express both FcγRI and II on their surfaces, were added and the samples were incubated at 37 °C under 5% CO₂. After 2 h, RPMI medium with 4% FBS was added. The cell–HuMAb–DENV solution was incubated for 3 days, after which RNA was extracted from the infected cells using TRIzol^® reagent (Invitrogen). The viral RNA was quantified by a one-step Realtime PCR using dengue virus specific primer and glyceraldehyde 3-phosphate dehydrogenase (GADPH) as an internal control (Sasaki et al., 2013). The result is presented as the fold enhancement of virus copies compared with the virus–PBS mixture (control).

Phage affinity selection (Biopanning)

Human monoclonal antibody clone B3B9 that show cross-neutralizing activity to 4 serotypes of DENV was used for mapping the epitope (Rowley, O’Connor & Wijeyewickrema, 2004). Panning was performed by using Ph.D.-12 and Ph.D.-C7C Phage Display Peptide Library Kit (New England Biolabs Inc., Hitchin, UK) according to the manufacturer’s instructions. As described in the manufacturer lab manual, the dodeca-and loop-constrained hepta- peptide library consists of 1.2 × 10⁹ electroporated sequences.

Briefly, 50 µl of protein A/G magnetic beads were blocked with BSA by incubating at room temperature for 1 h, and washed with 0.1% Tris Buffered Saline with Tween (TBST). In the meantime, Phages (5 × 10¹⁰ plaque forming units (PFU)) were incubated at room temperature for 30 min with purified B3B9 HuMAb to a final volume of 200 µl TBST. Phage—HuMAb mixture was transferred to the tube containing the blocked magnetic beads and incubated for 20 min at room temperature. After incubation, nonbinding phages that were not captured on magnetic beads were washed with TBST. The bound phages were eluted from magnetic beads with 1 ml glycine elution buffer (0.2 M Glycine-HCl, 1 mg/ml BSA, pH 2.2) and neutralized with 150 µl of 1M Tris–HCl (pH 9.1). Then, the bound phages were directly added to 20 ml early-log Escherichia coli (E. coli) ER2738 culture and incubated at 37 °C with vigorous shaking for 4.5 h. After incubation, the culture was transferred to a 50 ml centrifuge tube and spun at 5,000 rpm for 15 min at 4 °C. The phage-containing supernatant was transferred to a new 50 ml centrifuge tube. These amplified phages were precipitated with 20% PEG 8000/2.5 M NaCl at 4 °C overnight, and centrifuged at 12,000 g for 15 min. Phage pellets were then resuspended with 200 µl TBS and used in the next cycle. Three rounds of selection were performed. Specificity of selected phage clones were confirmed by phage ELISA. Briefly, 96-well microtiter plates were coated in triplicate well of 100 µl B3B9 HuMAb at 4 °C overnight. Then, the wells were washed two times with 0.05% Phosphate Buffered Saline with Tween (PBST). After that, the wells were blocked with blocking buffer (3% BSA in TBST). Then, phage lysate of each selected clones from panning step was added to two wells of coated antibody. At this step, wild type M13 phages were added to another HuMAb-coated well as a no fusion peptide control. Consequently, anti-M13 antibody-HRP conjugated; dilute 1:5,000 in blocking buffer were added. All incubations were performed in humidity chamber at 37 °C, for 1 h. After washing, the reaction was developed using 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) substrate (Sigma-Aldrich), and terminated by adding 100 µl of 2.0 M H₂SO₄. The absorbance was measured at 450 nm using ELISA reader (TECAN). Plasmids from positive clones of ELISA were isolated and sequenced using -96 gIII sequencing primer 5′-TGA GCG GAT AAC AAT TTC AC-3′. This primer was used to amplify the fusion peptide region. The inserted random 7 and 12 amino acids were identified and analyzed. All inserted sequences were aligned to determine the consensus sequence. The obtained consensus sequences were aligned with dengue viral genome to determine the mimic epitope.

Phage affinity binding by inhibition ELISA

After sequence analysis, inhibition ELISA was used to confirm candidate phages from each group of inserted peptide sequences that had specifically bound to B3B9 HuMAb. Reciprocal dilution of each phage clones were firstly determined to use in inhibition assay. For inhibition ELISA, B3B9 HuMAb was serially 2-fold diluted started at 20 µg/ml. Then, the diluted antibody was incubated with equal volume of phage at specified dilution at room temperature for 1 h. After incubation, the mixtures were transferred to HuMAb pre-coated plate, and incubated for another 1 h. Bound phages to the coated HuMAb were detected by anti-M13-HRP, and signal was developed with TMB substrate. The absorbance values were measured at 450 nm using an ELISA reader (TECAN). Absorbance values of each concentration (A) of antibody in solution phase were divided by absorbance values in the absence of antibody (A0), yielding normalized values (A/A0) (Skottrup et al., 2012). Non-relevant anti-influenza antibody, which was tested as negative binding with our selected dengue-specific phage clones, was used as a negative control.

Wild type and mutated human monoclonal antibody clone B3B9 were generated and showed cross reactivity to all serotypes of DENV

The substitution of amino acid at position 297 from Asparagine to glutamine was created and confirmed by DNA sequencing. Five mg of wild type B3B9 and four mg of N297Q-B3B9 HuMAb were obtained. The successful production of wild type and mutant (N297Q) rIgG was verified by the capacity of binding activity observed via IFA. The N297Q-B3B9 rIgG displayed cross-reactivity to all four DENV serotypes similarly to the wild type rIgG (Figs. 1A–1D).

Human monoclonal antibody clone B3B9 is IgG1 isotype

Reverse transcription PCR was performed to determine the B3B9 rIgG isotype. Using B3B9 hybridoma complementary DNA as template, the PCR product with approximately 211 bp of IgG1 was observed (Fig. S1).

Wild type and mutated human monoclonal antibody clone B3B9 showed cross neutralizing activity against all serotypes of DENV

To determine the neutralizing activity of rIgG, we assessed the NT of N297Q-B3B9 rIgG and B3B9 rIgG in Vero cells. N297Q-B3B9 rIgG displayed almost the same level of NT as the B3B9 rIgG against all DENV serotypes. The NT levels of various concentrations of these two antibodies against all four DENV serotypes are shown in Figs. 2A–2D. Among these four serotypes, B3B9 and N297Q-B3B9 rIgG showed identical 50% reduction of FFU (NT50) to DENV-2, 3, and 4 (0.125 µg/ml for DENV2 and 2 µg/ml for both DENV3 and DENV4). However, the NT50 concentration against DENV1 of N297Q-B3B9 rIgG was slightly higher than B3B9 rIgG (Fig. 2E).

Mutated human monoclonal antibody clone B3B9 eliminated ADE activity in K562 cells while wild type showed ADE activity against all types of DENV

B3B9 rIgG was able to neutralize DENV2 and 3 at the minimum concentration, 0.39 and 25 µg/ml respectively (Figs. 3B–3C), but not neutralize to DENV1 (Figs. 3A) and 4 (Fig. 3D). Moreover, this antibody induced a virus infection enhancement at concentrations 400–0.0015 µg/ml, 0.39–0.006 µg/ml, 100–0.0015 µg/ml, and 400–0.00038 µg/ml for DENV1, 2, 3, and 4, respectively (Figs. 3A–3D). To the contrary, N297Q-B3B9 rIgG showed NT for all four DENV serotypes and showed no enhancing activity in any of the tested antibody concentrations.

Mutated human monoclonal antibody clone B3B9 showed no ADE activity in THP-1 cells while wild type showed ADE activity against all types of DENV

In THP-1 cells, mutant IgG (N297Q-B3B9) that cannot bind to FcγR showed a complete reduction of ADE activity in all tested antibody concentrations. In contrast, wild type rIgG induced 9541, 43, 3061, and 2020 fold enhancement in the dengue virus RNA copy number compared with the control for DENV1, 2, 3, and 4, respectively (Figs. 3E–3H).