Dissecting antibody-dependent enhancement modulation by Fc-modified cross-neutralizing human monoclonal antibody

Virus and cells

The DENV1 Mochizuki strain, DENV2 New Guinea C (NGC) strain, DENV3 H87 strain, and DENV4 H241 strain, kindly provided by Prof. Kazuyoshi Ikuta, Research Institute for Microbial Diseases (RIMD), Osaka University, were propagated in C6/36 cells cultured in Leibovitz L15 medium (Hyclone) with 10% fetal bovine serum (FBS) (Hyclone) and 0.3% tryptose phosphate broth. Vero cells were obtained from Prof. Kazuyoshi Ikuta, RIMD, and used for the neutralization activity test. The cells were maintained in the minimal essential medium (MEM) (GE Healthcare UK Ltd., Buckinghamshire, UK) with 10% FBS. For the ADE test, K562 cells were kindly provided by Assoc. Prof. Atsushi Yamanaka, RIMD, and cultured in the RPMI 1640 medium (Hyclone) supplemented with 10% FBS (Hyclone^®, USA). CHO-K1 cells were obtained from Assoc. Prof. Atsushi Yamanaka, RIMD, were used for the stable expression of B3B9, N297Q-B3B9, and LALA-B3B9 antibodies. These cells were cultured in the MEM medium supplemented with 10% FBS and 1% nonessential amino acids (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Portions of this text were previously published as part of a preprint (https://www.biorxiv.org/content/10.1101/2023.05.25.542225v1.full).

Generation of LALA-mutated human monoclonal antibody clone B3B9

Transient expression of LALA-B3B9 mutation antibody

After sequencing of the mutated heavy chain plasmid for confirmation, the heavy- and light-chain plasmids were transfected to 6.8 × 10⁶ HEK293T cells in a T75 cell culture flask for transient expression. The culture medium was collected and used in immunofluorescence assays to determine the DENV-binding activity. The culture medium containing the secreted LALA-B3B9 recombinant immunoglobulin G (rIgG) was purified using the protein A affinity column (GE Healthcare, Chicago, IL, USA). The eluted fractions from purification were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the purity of the purified antibody. The LALA-B3B9 rIgG concentration was measured using the BCA protein assay kit (Thermo Scientific, Waltham, MA, USA).

Indirect immunofluorescence assay for the DENV-binding activity

Indirect immunofluorescence assay was used to assess the binding activity of the LALA-B3B9 antibodies for all DENV serotypes. Vero cells were mock-infected or infected with DENV at a multiplicity of infection (MOI) of 0.1 in 96-well cell culture plates for 72 h. After fixing the cells with 3.7% formaldehyde in phosphate-buffered saline (PBS) and permeabilized with 0.1% Triton X-100 in PBS, the cells were stained with culture supernatant from transfected cells as primary antibody. After incubation at 37 °C for 1 h, Alexa Fluor 488-conjugated anti-human IgG (Thermo Fisher Scientific. Invitrogen. Waltham, MA, USA.) (1:1,000) was added as a secondary antibody. The fluorescence can be visualized using a fluorescence microscope (IX71; Olympus, Tokyo, Japan).

Cellular binding activity of B3B9, N297Q-B3B9 and LALA-B3B9 mAbs to CD32a (FcγRIIa, H131)

The Tag-lite binding assay was used to evaluate the binding activity of the Fc domain of the B3B9, N297Q-B3B9, and LALA-B3B9 monoclonal antibodies with FcγRIIa (CD32a) (Cisbio, Codolet, France). In this assay, binding activity of unlabeled antibodies to the receptor were determined by competing with an acceptor labeled human IgG (IgG-d2). HEK293 cells expressing the FcγIIA receptor labeled with Lumi4-terbium Cryptate (Lumi4-Tb) were dispensed, followed by the addition five µl of IgG1 labeled with d2 and five µl of the unlabeled sample (B3B9, N297Q-B3B9, or LALA-B3B9 mAb) in a concentration ranging from 3–5 nM. After 2 h of incubation at room temperature, the signal was measured at 620 nm for the fluorescent of Lumi4-Tb and at 665 nm for the fluorescent acceptor dye (d2). A standard curve was generated from a known concentration of IgG1 Protein (Degorce et al., 2009). Data obtained were compared with the serum-free media used as a negative control and human IgG1 protein (D239E, L241M, HEK293) (MedChemExpress), which is the reference recombinant human-derived IgG1 protein expressed by HEK293 and interacts with the Fc gamma receptor IIa, used as the positive control.

Foci reduction neutralization test of LALA-B3B9 antibodies

The neutralization activity of the LALA-B3B9 antibodies was assessed and compared with that of the previously generated aglycosylated HuMAb (N297Q-B3B9) (Injampa et al., 2017) and wild-type antibody (B3B9) using Vero cells, as described in a previous study (Pitaksajjakul et al., 2014). In brief, DENV at an MOI of 0.01 was preincubated with the respective purified LALA-B3B9 or N297Q-B3B9 or B3B9 HuMAb at 37 °C for 1 h before infecting Vero cells in 96-well-cell culture plates. At 2 h post-infection, the plate was overlaid with 2% carboxymethyl cellulose in the MEM medium with 2% FBS. The cells were incubated for 2 days for DENV4 and for 3 days for DENV1, DENV2, and DENV3. Then, the cells were fixed and immune-stained using anti-DENV human antibody, followed by Alexa-conjugated anti-human IgG (H+L) (1:1,000 dilution). Neutralization activity was quantified by counting the number of foci for each antibody concentration under a fluorescence microscope compared with the negative control (absence of antibody). The neutralizing antibody titer was considered the minimum IgG concentration yielding a 50% reduction in the focus number (FRNT50). In this study, the anti-influenza virus HuMAb (5E4) was used as an unrelated control.

Antibody-dependent enhancement assay of LALA-B3B9 antibody

The ADE activity of the LALA-B3B9 antibody was assessed using semi-adherent FcγRIIa-bearing K562 cells and compared with that of N297Q-B3B9 and B3B9 (Konishi, Tabuchi & Yamanaka, 2010). In total, 36 microliters of serially diluted antibodies were mixed with 50 µl of DENV in 10% FBS RPMI medium in 96-well poly-L-lysine-coated plates (Corning Inc., NY, USA). The mixture was incubated for 2 h at 37 °C, supplied with 5% CO₂. The mixture was then added with 50 µl K562 cells at a density of 2 × 10⁶ cells/ml and incubated at 37 °C under 5% CO₂ for 2 days. Thereafter, the cells were washed with PBS three times, dried, and fixed with an acetone/methanol fixing solution at −20 °C for at least 30 mins, and immune-stained with D23–3E6D7, an in-house anti-DENV human antibody specific to E protein (Setthapramote et al., 2012), followed by Horseradish peroxidase-conjugated anti-human IgG (H+L) (Sigma-Aldrich, St. Louis, MO, USA). The signal was developed with a DAB substrate solution (KPL, Gaithersburg, MD, USA). The ADE activity was measured by counting virally infected cells manually under a light microscope at a magnification of 10x. Stained cells were counted in three random fields using a 10 × 10 GRID Microscope Eyepiece Micrometer. The total infected cell count in a well was calculated from the average number of stained cells in one grid multiplied by 153.86 (the area of one well was 153.86 times greater than grid square). The cut-off value for distinguishing enhancing from non-enhancing activities was calculated from the percentage of infected cells obtained using six negative controls. This value represents the number of infected cells when the virus was added to cells without antibody, tested in triplicate assays within each ADE experiment, with the experiments performed in two independent replicates. The mean percentage of infected cells from these negative controls was used to calculate the cut-off value, defined as the mean ± one standard deviation (mean ± SD). Antibody concentrations resulting in a higher or lower number of infected cells compared with the cut-off value were considered indicative of ADE or neutralization, respectively.

Stable expression of B3B9, N297Q-B3B9, and LALA-B3B9 antibodies

The generation of a stable antibody-secreting cell line was described in a previous study (Injampa et al., 2017). Briefly, CHO-K1 cells were seeded at 1.3 × 10⁶ cells in a six-well-cell culture plate. The constructed plasmids expressing the heavy and light-chains, which contained puromycin- and hygromycin-resistant genes, respectively, were co-transfected into the cells using transfection reagent (Lipofectamine^® 2000; Thermo Fisher Scientific, Waltham, MA, USA). After 2 days, the culture medium was replaced with MEM supplemented with 10% FBS and 1% NEAA containing puromycin and hygromycin at eight and 800 µg/ml, respectively. After 7 days, when all cells in the control well without plasmid had died, the cells that survived under antibiotic selection were expanded and stored as stable pool. Some cells were used for cloning by limiting dilution in 96 well-cell culture plates with selection media. The selected single-colony clones that tested positive for dengue virus binding by IFA were scaled up for subsequent antibody preparation and characterization.

Complement-induced antibody-dependent activity assay

Serial antibody dilutions were incubated with DENV with or without rabbit complement (Cedarlane, Ontario, Canada) for 2 h at 37 °C. The mixture was mixed with 50 µl of 2 × 10⁶ K562 cells/ml and incubated for 2 more days, followed by fixation and immunostaining of the cells. To differentiate enhancing from neutralizing activities, the cut-off value was determined using the percentage of infected cells measured in six negative controls. These controls—where virus was added to cells without antibody—were tested in triplicate within each assay, and the entire experiment was conducted in two independent replicates. The cut-off value was calculated as the mean percentage of infected cells from the mean ± one standard deviation (mean ± SD). Antibody concentrations resulting in infected cell percentages above or below this cut-off were interpreted as indicative of ADE or neutralization, respectively (Yamanaka, Kosugi & Konishi, 2008).

In vitro suppression-of-enhancement assay in K562 cells

In vitro suppression-of-enhancement assay

First, the optimal serum dilution for DENV2 infected patient showing the greatest enhancement of distinct dengue virus serotypes in K562 cells was determined (1/4,000 serum dilution for DENV1 and DENV2, 1/1,000 serum dilution for DENV3 and DENV4). The patient serum at the predetermined concentration was then mixed with DENV at an MOI of 0.1 and incubated for 1 h at 37 ^∘C with 5% CO₂ prior to the addition of two-fold serially diluted HuMAbs starting at 1,000 μg/ml. After incubation for 1 h at 37 °C, 50 µl of 2 × 10⁶ cells/ml K562 cells were added to the serum–virus–antibody mixture. At 48 h post-infection at 37 °C with 5% CO₂, the cells were fixed by acetone/methanol fixing solution at −20 °C, and immunostaining for the viral antigen was performed by incubating with D23–3E6D7, an in-house anti-DENV human antibody (Setthapramote et al., 2012) for overnight at 4 °C. After incubation, the plate was washed three times and incubated with Horseradish peroxidase-conjugated antihuman IgG (H+L) (Sigma-Aldrich, St. Louis, MO, USA) diluted in 0.05% Tween-20 and 1% FBS in PBS for 1 h at 37 °C. To visualize the infected cells, the signal was developed with a DAB substrate solution (KPL, Gaithersburg, MD, USA). The cut-off value to distinguish enhancing from neutralizing activities was based on the percentage of infected cells in six negative controls, where virus was added without antibody. These were tested in triplicate across two independent experiments. The cut-off was set as the mean ± one standard deviation (mean ± SD) (Williams et al., 2013).

Statistical analysis

All results were expressed as mean ± SD. All calculations were performed using the GraphPad Prism 6 software.

Ethical statement

All experimental procedures using human samples were preapproved by the Ethics Committee of the Faculty of Tropical Medicine (FTM), Mahidol University (protocol number: fTM ECF-019-05). All donors provided a written informed consent before enrollment.

Generation and cross-reactivity of the Fc-modified LALA-B3B9 mutation antibody

The substitution of amino acids at positions 234 and 235 from leucine to alanine in the Fc portion of monoclonal antibody B3B9 was confirmed by DNA sequencing. The cross-reactivity of the LALA-B3B9 antibodies to all DENV serotypes was determined using an immunofluorescence assay and compared with a negative control of mock-infected cells. Results showed that LALA-B3B9 antibodies reacted with all DENV serotypes (Fig. 1A). The purity of the produced LALA-B3B9 antibody was assessed by western blot analysis, which detected a specific IgG band at approximately 250 kDa under non-reducing conditions (Fig. 1B).

Cellular binding activity of B3B9, N297Q-B3B9 and LALA-B3B9 mAbs to CD32a (FcγRIIa, H131)

The binding activity of the Fc portion of the generated antibodies with the Fc receptor was measured using a competition assay format, utilizing the cell surface bound FcγIIa receptor and the antibody variant. The result in Fig. 2. revealed that the B3B9 antibody effectively competes with acceptor-labeled human IgG for binding with FcγRIIa, resulting in a decrease fluorescence signal at every antibody dilution compared to the negative control (serum-free medium). In contrast, LALA-B3B9 and N297Q-B3B9 demonstrated similar binding behavior, with no significant difference in the fluorescent signal compared to the negative control. Furthermore, these antibodies exhibited significantly lower binding capability to the Fc portion than the positive control protein (Human IgG1). These results suggest that the two Fc modified antibodies diminished the binding between their antibody Fc and FcγRIIa.

Neutralization of all dengue virus serotypes by the Fc-modified LALA-B3B9, N297Q-B3B9 and B3B9 antibody

The FRNT50 test was used to assess the capabilities of LALA-B3B9 HuMAbs to neutralize DENV1–4 (Arduin et al., 2015). The neutralizing abilities of the HuMAbs were compared among B3B9, wild-type antibody, and N297Q-B3B9 variant, an Fc-modified antibody with a substitution at position N297Q, as described in Fig. 3. All HuMAbs were cross-reactive with four serotypes of dengue virus with different neutralizing potency. At the highest concentration of 64 μg/ml, all HuMAbs reduced DENV2 and DENV4 by 100% and DENV3 by nearly 90%. For DENV1, LALA-B3B9, N297Q-B3B9, and B3B9 antibodies did not achieve 100% neutralization. The FRNT50 values of the three generated antibodies are shown in Table 1. These values differed significantly for dengue serotypes 1, 3, and 4 (p < 0.05), indicating significant differences in neutralization potency among the antibodies. In contrast, no significant differences were observed among the antibodies for dengue serotype 2 (p > 0.05). In terms of neutralization potency, the LALA-B3B9 antibody exhibits moderate activity against DENV2 and DENV4, with 50% neutralization titers ranging from 0.1 to 1 µg/ml., with FRNT50 values at 0.79 (±0.27) and 0.8 (±0.265) µg/ml, respectively. For DENV3, the FRNT50 concentrations of the LALA-B3B9 antibody was 4.97 (±0.535) µg/ml. The FRNT50 concentrations of N297Q-B3B9 against DENV1 and DENV3 were lower than those of LALA-B3B9 and B3B9 HuMAb but higher against DENV4 (p < 0.05).

ADE activity of the Fc-modified B3B9 LALA mutation antibody

The higher infection rates of immune effector cells occur via the ADE phenomenon, which is mediated by Fc–FcR interactions. Hence, not only neutralizing activity but also ADE activity is a substantial concern for antibody-based therapeutic development. In this study, the ADE activity was determined using FcγRII-bearing K562 cells. Figure 4 shows the comparison between wild-type antibody (B3B9), aglycosylated antibody (N297Q-B3B9), and Fc-modified antibody (LALA-B3B9). The neutralizing and enhancing activities were assessed by comparing the number of infected cells at each antibody concentration to the control without antibody. The results showed that ADE activity was completely abolished for the Fc-modified antibodies N297Q-B3B9 and LALA-B3B9 across all antibody concentrations. At higher concentrations, these variants effectively neutralize all dengue virus serotypes. In contrast, the B3B9 antibody exhibited weak neutralizing activity, particularly against DENV1, and induced ADE at sub-neutralizing concentrations across all serotypes.

Complement-induced antibody-dependent activity assay

Since complement can play a role in the protective activities against viral infection, Fc modification not only reduces Fc receptor binding but also decreases binding to the C1q complement protein. Therefore, the balance between the enhancing and neutralizing activities of both types of modified antibodies and the wild-type antibody were investigated using the complement-induced antibody-dependent activity assay (Yamanaka & Konishi, 2017). For the wild-type antibody, complement proteins helped reduce ADE activity across all dengue virus serotypes, particularly DENV1 and DENV2, where ADE was significantly abolished. The number of infected cells during ADE activity in the presence of complement was significantly lower than those in the absence of complement, at antibody concentrations of 1.56–25 µg/ml for DENV1, 0.097–1.56 µg/ml for DENV2, 0.39–25 µg/ml for DENV3, and 0.006–400 µg/ml for DENV4 (Fig. 5A). However, despite the presence of complement, enhancing activity was still observed against DENV3 at antibody concentrations higher than 0.024 µg/ml and against DENV4 at all concentrations.

In contrast, for the Fc-modified antibodies, there was no significant difference in the neutralization potency of LALA-B3B9 and N297Q-B3B9 HuMAbs in the presence or absence of complement proteins (Figs. 5B, 5C). These antibodies can neutralize all four DENV serotypes without enhancing infectivity. Therefore, both the neutralizing and enhancing activities of N297Q-B3B9 and LALA-B3B9 were complement-independent.

In vitro suppression-of-enhancement assay in K562 cells

The serum samples of patients with acute DENV-2 infection at enhancing concentrations were used to predict the competitive ability of the Fc-modified antibodies and wild-type antibodies to natural dengue infection. The use of human serum samples was approved by the Ethical Committees of the Faculty of Tropical Medicine, Mahidol University. The number of infected cells was compared with that of controls (the mean number of cells infected by a mixture of virus and patient serum without antibody ±SD) to determine the neutralizing or enhancing activity. Each generated antibody dilution was mixed with the DENV2 serum at an enhancing concentration that pre-incubated with virus. Then, K562 cells were added. LALA-B3B9 and N297Q-B3B9 exhibited neutralizing activity against all virus serotypes without enhancing their infectivity. There were no differences in the neutralization and enhancement activity of the two Fc-modified antibodies (p > 0.05).

The B3B9 antibody showed a higher number of infected cells than those from both the no antibody control and Fc-modified antibodies for DENV1 –4, at antibody concentration ranges of 15.625-1000, 1.95–62.5, 1.95-250 and 1.95 –31.25 µg/ml, respectively. This antibody presented with enhancing activity against DENV1 at antibody concentrations of 500–15.63 µg/ml and DENV3 at antibody concentrations of above 31.25 µg/ml. However, it was able to neutralize DENV2 and DENV4 at all antibody dilutions (Fig. 6).