Monoclonal antibody against EV71 3D^pol inhibits the polymerase activity of RdRp and virus replication

Generation of mAbs against 3D^pol of EV71

We generated mAbs against 3D^pol by traditional hybridoma method and obtained a panel of mAbs against EV71 3D^pol. We achieved three mAbs 3A12, 2A10 and 7A6G1, and the property of each mAb was summarized in Table 1. 3A12 and 7A6G1 are IgG1 subtype with typical heavy chains about 50 kD and light chains about 27 kD, while 2A10 is IgG2a subtype with typical heavy chains about 50 kD and light chains about 24 kD (Fig. 1a). Ratio of each mAb heavy chain(HC)/light chain(LC) was close to 2 (Additional file 1: Figure S1). Specificities of the mAbs were identified with EV71-infected Vero-1008 cells by IFA using the mAbs as primary antibodies. The serum from 3D^pol-immunized mice was used as positive control here. The mAb 5G10 which is specific to Salmonella flagellin severed as negative mAb control [11]. The result showed the EV71-specific green florescent signals were mostly located in cytoplasm (Fig. 1b). Mean fluorescence intensity (MFI) of each virus infected cell was about 5000 (Additional file 2: Figure S2). Cell lysate of EV71-infected Vero-1008 cell was subject to Western blotting assay, a clear 51 kD band of 3D^pol and a 55 kD band of cleaved 3CD^pol protein could be detected, while a 70 kD band of 3CD^pol (a precursor of 3D^pol) could also be detected (Fig. 1c) as similar as reported previously [12].

mAbs	IgG subtype	Recognized region (AA)	ELISA	IFA	Western blot
3A12	IgG1	1–120	+++	+++	++
2A10	IgG2a	121–250	+++	+++	++
7A6G1	IgG1	360–462	+++	+++	+

AA amino acid

Identification of the regions recognized by the mAbs

To identify the recognized epitopes recognized by the distinct mAbs, three recombinant 3D^pol proteins with serial C-terminal truncations were prepared for Western blotting assay (Fig. 2a). 3A12 showed binding ability to all the three truncated proteins and the intact 3D^pol protein. 2A10 showed binding ability to all the proteins but truncated in C-terminal 120–462 amino acid, while 7A6G1 to none of the three truncated proteins but only the intact 3D^pol (Fig. 2b). The result suggested that the 3A12 recognized 3D^pol in 1–120 amino acid of 3D^pol N-terminal, 2A10 recognized in 120–250 amino acid, and 7A6G1 recognized in C-terminal 360–462 amino acid (Fig. 2c).

In vitro inhibition abilities of the mAbs

Inhibition abilities of the mAbs were rapidly evaluated in an RdRp-mediated RNA elongation system (Fig. 3a), in which antibody binds to RdRp, the RNA elongation would be blocked. When mAbs 3A12 or 2A10 engaged with 3D^pol, the elongated RNA band almost disappeared, however the applications of 7A6G1 and 5G10 did not significantly inhibit the quantity of elongated RNA (Fig. 3b). 5G10 served as an irrelative antibody control. Therefore, mAbs 3A12 and 2A10 remarkably inhibited the RNA elongation compared with 7A6G1 and 5G10.

Antiviral capacity of the intracellular mAbs

In order to demonstrate whether intracellular mAbs could inhibit viral replication, antiviral capacity of mAbs were evaluated in EV71-infected cell. It was observed that over 80% cell were transfected with mouse IgG upon DOTAP (data not shown). After transfection with mAbs (3A12, 2A10, 7A6G1 or 5G10), EV71-infected cells were taken photographs at 24 h, it was observed that 3A12 and 2A10 resulted in less CPE when compared with 7A6G1 and 5G10 (Fig. 4a). And the EV71-infected samples were subjected to test the viral titer. Among those applied antibodies, 3A12 or 2A10-transfected Vero-1008 produced about 2000 PFU of EV71 per well, which was significantly lower than 7A6G1 or 5G10-transfected cells (p < 0.01) (Fig. 4b). However, without the application of DOTAP both the 3A12 and 2A10 could not suppress the production of EV71 (Fig. 4b).

Furthermore, it was demonstrated whether antibody-mediated intracellular inhibition is dose-dependent. Based on the same method, EV71-infected Vero-1008 were transfected with mAbs 2A10 or 5G10 at increasing dose, subsequently the viral production decreased accompanying the increasing quantity of mAb 2A10 (Fig. 4c). Without transfection process, 3A12 and 2A10 could almost not interrupt viral propagation (Additional file 3: Figure S3). In summary, 3A12 and 2A10 intracellularly neutralize 3D^pol and inhibit viral replication in a dose-dependent manner.

Our IFA results also showed that majority of 3D^pol proteins (RdRp) located in cytoplasm and nuclear of EV71-infected cells (Fig. 1b). Extracellular 3A12 and 2A10 with molecular weight about 150 kD were barely able to pass through the cell membrane into cytoplasm in vitro. Only by intracellularly delivering by using protein transfection reagent DOTAP could 3D^pol-specific mAbs exert antiviral function.

An in vivo evaluation of antiviral efficacy of the mAbs

To identify whether the 3D^pol-specific mAbs could exert antiviral function in vivo, neonatal ICR mice i.p. received mAbs and were then challenged as described in Methods. Briefly, five ICR mice with 12 days’ pregnancy were purchased and housed individually. At that time, each pregnant mouse was randomly allocated to different treatment groups (3A12, 2A10, 7A6G3, 5G10, and PBS) in a single-blind manner. Subsequently, 13, 11, 11, 9, and 10 neonatal mice were given birth in the corresponding broods respectively. Therefore 54 neonatal mice were totally delivered and included in the in vivo study. After challenge, all the neonatal mice in control group develop typical EV71 clinical syndrome including limb weakness and paralysis (Fig. 5a and b) which were calculated in a double-blind manner as previously described [11]. After observation for 16 days, five and three mice still survived in those groups received injection of 3A12 and 2A10, respectively. Thus 3A12 (5/13, 38.5%) and 2A10 (3/11, 27.3%) provided significantly higher protection efficacy then 5G10 (0/9, 0%) (p < 0.01) (Fig. 5c). Interestingly, although 7A6G1 finally did not confer protection for mice, it prolonged the survival time for the challenged mice.

Cell, virus, antibody and protein

Vero-1008 was grown and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, MO, USA) supplemented with 10% heated-inactivated fetal bovine serum (Gibco, Life Technologies Corp., Calif., USA), 100 μg/ml streptomycin and 100 IU/ml penicillin (Sigma-Aldrich, MO, USA) at 37 °C with 5% CO₂. A BrCr strain of EV71 was provided by Dr. H Wang in Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS). A mouse-adapted EV71 (EV71/MAV-VR) used in this study was kindly provided by Dr. Z Huang in Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, CAS, China [21]. The BrCr and MAV-VR EV71 strains have the same 3D^pol amino acid sequence. For clarification, EV71/MAV-VR was only used in the challenge experiment, while the rest EV71 mentioned in this study represent EV71 BrCr strain. A purified recombinant 3D^pol protein was provided by Dr. P Gong in WIV, CAS [22].

Animals

BALB/c aged 6–8 wks and pregnant ICR mice were purchased from Beijing Laboratory Animal Research Center and housed under specific pathogen free (SPF) conditions in the Animal Center of WIV, CAS, China.

Generation of mAbs against EV71 3D^pol

IgG mAbs against EV71 3D^pol were developed by traditional hybridoma technique as previously described [23]. In brief, 5-wk-old female SPF BALB/c mice were immunized subcutaneously with 100 μg of 3D^pol at 2-wk interval. Four wks after the last booster and 3 days before cell fusion, the mice were boosted with 200 μg of 3D^pol. Three days later, murine splenocytes were harvested and fused with SP2/0 using 50% polyethyleneglycol (Sigma-Aldrich, MO, USA). Hybridoma culture supernatants were preliminarily screened by EV71 3D^pol protein using ELISA. The positive hybridoma cells were cloned by a limiting dilution and the stable hybridoma clones were injected into liquid paraffin-pretreated abdominal cavities of BALB/c mice. Subsequently, the mAbs were harvested and purified from the ascite with an antibody purification kit according to the manufacturer’s specifications (NAb™ Protein A/G Spin Kit, Thermo Scientific, IL, USA). This mouse study was approved by the ethics committee of life science and research in Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS) (No. WIVA09201502).

Immunofluorescence assay (IFA)

Vero-1008 cells were seeded into 24-well tissue culture plates (Costar Corning Inc., NY, USA) at a concentration of 1 × 10⁵ cells per well. When the cells reached approximately 80% confluence, culture medium was removed and then cells were washed three times with sterile PBS (pH 7.4) and incubated with EV71 (MOI = 0.1) for 1 h at 37 °C. After removal of supernatant, fresh medium was added and cultures were incubated at 37 °C. At 24 h of post infection, the infected cells were fixed with absolute methanol and processed for indirect immunofluorescence assay (IFA) using purified mAbs, followed by fluorescein isocyanate-conjugated goat anti-mouse IgG for confirming antibody specificity against EV71. Fluorescent images were examined under a fluorescent microscope.

Immunoblotting

The EV71-infected Vero-1008 were separated by SDS-PAGE and then electrophoretically transferred to PVDF membrane (GE Healthcare, PA, USA). The membrane was blocked for 1 h at RT with blocking solutions containing 5% Bovine Serum Albulmin (BSA) in TBS (20 mM Tris-HCl (pH 7.5), 150 mM NaCl), and then incubated overnight with purified mAbs at 4 °C. After washing with T-TBS (TBS + 0.05% Tween 20), the membrane was incubated for 45 min with HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, PA, USA). After washing with T-TBS, the membrane was developed by treatment with ECL Western Blotting Detection Reagents (Boster, Wuhan, China). A house keeping protein β-actin was also detected as control.

EV71 infection and mAbs IgG transfection

Vero-1008 cell was seed in 24-well plate 1 × 10⁵ cell per well culturing for 24 h, and was infected with EV71 (MOI = 0.1). Three hrs later, EV71-infected were transfected with IgG using Liposomal Transfection Reagent DOTAP (Roche, Basel, Switzerland) as previously described [24]. Briefly, 15 μl of DOTAP was incubated with 10 μl of purified IgG (5 μg/well) for 20 min in serum-free medium. After incubation, 500 μl DMEM medium and mixture was added. Twenty-four hrs later, virus-infected cell were taken pictures and undergone three freeze-thaw circles, and were subject to determine the viral titers.

Plaque assay

A series of 1:10 dilutions were made by mixing 15 μl of EV71-infected cell with 135 μl of DMEM. One hundred microliters of each dilution were seeded to individual wells of 24-well plate containing confluent Vero-1008 cells (2 × 10⁵ cells/well). The plate was incubated at 37 °C with 5% CO₂ for 1 h before the layer of 2% methyl cellulose was added. After 4 days of incubation at 37 °C with 5% CO₂, the cells were fixed in 3.7% formaldehyde and then stained with 1% crystal violet. Plaque numbers were recorded after washing the plates with tap water [25].

RdRp-mediated RNA elongation assay in vitro

In 10 μl reaction system containing 50 mM HEPES, 75 mM KCl, 5 mM MgCl₂, 4 mM TCEP, 300 μM NTP, and 4 μM RNA Complex, the 5 μg functional 3D^pol was added, and the system maintained at 22.5 °C for 30 min, finally the reaction was terminated with RAB. RNA species were resolved by 15% polyacrylamide/7 M Urea gel electrophoresis and visualized by Stains-All staining [26]. RNA elongation activity of 3D^pol could be assessed based on the appearance of elongated RNA band.

In vivo evaluation of antiviral efficacy of mAbs

The protective efficacy of the mAbs was evaluated by an in vivo assay as previously described [21]. Pregnant ICR mice maintain under SFP condition. Five groups of neonatal ICR mice at 1 day of age were inoculated i.p. with 50 μg of mAb/mouse (50 μl) respectively, followed by i.p. inoculation with 3.55 × 10⁶ TCID₅₀ of EV71/MAV-VR (50 μl) at 24 h later. The challenged mice were monitored daily for survival and clinical score for 16 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; and 4, death. Mice that lost more than 35% of the control mice (PBS treated group) body weight were euthanized and counted as dead. All the mice were observed for 16 days and euthanized by injection with ketamine mixture. Mice were euthanized by injection (i.m.) with 15 μg/50 μl of ketamine mixture (79.3% Ketaject, 17.5% Atropine, and 3.2% Acepromazine) per gram mice body weight. The EV71/MAV-VR challenge study was approved by the ethics committee of life science and research in Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS) (No. WIVA09201504).

Statistical analyses

Kaplan-Meier survival curves were used to display mortality data, and log rank analyses were performed to determine statistical significance between different groups. * indicates value of p < 0.05, ** indicates value of p < 0.01.