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Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning Na Dong a,1 , Longlong Luo b,1 , Junhua Wu a , Peiyuan Jia a , Qian Li a,b , Yuxia Wang a,∗ , Zhongcai Gao a , Hui Peng b , Ming Lv b , Chunqian Huang a , Jiannan Feng b,∗∗ , Hua Li a , Junjie Shan a , Gang Han a , Beifen Shen b a b

Beijing Institute of Pharmacology and Toxicology, State Key Laboratory of Toxicology and Medical Countermeasures, Beijing 100850, China Beijing Institute of Basic Medical Sciences, Beijing 100850, China

a r t i c l e

i n f o

Article history: Received 27 March 2015 Received in revised form 15 June 2015 Accepted 19 June 2015 Available online xxx Keywords: Ricin Bioterrorism Monoclonal antibody

a b s t r a c t Ricin is a glycoprotein produced in castor seeds and consists of two polypeptide chains named Ricin Toxin A Chain (RTA) and Ricin Toxin B Chain (RTB), linked via a disulfide bridge. Due to its high toxicity, ricin is regarded as a high terrorist risk for the public. However, antibodies can play a pivotal role in neutralizing the toxin. In this research, the anti-toxicant effect of mAb 4C13, a monoclonal antibody (mAb) established using detoxicated ricin as the immunized antigen, was evaluated. Compared with mAb 4F2 and mAb 5G6, the effective mechanism of mAb 4C13 was analyzed by experiments relating to its cytotoxicity, epitope on ricin, binding kinetics with the toxin, its blockage on the protein synthesis inhibition induced by ricin and the intracelluar tracing of its complex with ricin. Our result indicated that mAb 4C13 could recognize and bind to RTA, RTB and exert its high affinity to the holotoxin. Both cytotoxicity and animal toxicity of ricin were well blocked by pre-incubating the toxin with mAb 4C13. By intravenous injection, mAb 4C13 could rescue the mouse intraperitoneally (ip) injected with a lethal dose of ricin (20 ␮g/kg) even at 6 h after the intoxication and its efficacy was dependent on its dosage. This research indicated that mAb 4C13 could be an excellent candidate for therapeutic antibodies. Its potent antitoxic efficiency was related to its recognition on the specific epitope with very high affinity and its blockage of protein synthesis inhibition in cytoplasm followed by cellular internalization with ricin. © 2015 Published by Elsevier Ltd.

1. Introduction Ricin is a heterodimeric ribosome inactivating protein which inhibits protein synthesis, consequently resulting in cell death [1,2]. It consists of two polypeptide chains named Ricin Toxin A Chain (RTA) and Ricin Toxin B Chain (RTB), linked via a disulfide bridge [3]. RTB mediates the binding to glycolipids or glycoproteins on the cell surface via its lectin receptors, followed by endocytic uptake of ricin into the cell [4]. After endocytosis, ricin is transported retrogradely from endosomes to the Golgi, and further on to the endoplasmic

Abbreviations: ip, intraperitoneal(ly); iv, intravenous(ly); mAb, monoclonal antibody; ELISA, enzyme linked immunosorbent assay; RTA, Ricin Toxin A Chain; RTB, Ricin Toxin B Chain. ∗ Corresponding author. Tel.: +86 10 66931645. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (J. Feng). 1 Equally contributed to the work.

reticulum (ER) [5]. As a potent toxin, ricin kills eukaryotic cells by inhibiting protein synthesis [6]. Ricin has been considered a potential agent of biological warfare or terrorist attack [7,8]. Because ricin inhibits protein synthesis very quickly, the cell or tissue damage begins within several hours although signs of intoxication might not be noted, making treatment difficult. Symptomatic therapies are likely to be effective in case of intoxication, but supportive measures are not so effective in rescuing the lives of people who have received a lethal dose of toxin. Therefore, attempts are being made to discover drugs that compete with or block the toxin from binding to its site of action. Both antibodies and competitive ligands inhibited binding of the toxin to cell membranes [9–12]. Ricin intoxication could be prevented by active immunity with toxoid [13,14], or by passive immunization with antibodies against the toxin [15–17]. Vaccine immunization and antibody prophylaxis will be effective only when the exact time of terrorist attack is known. However, passive immunotherapy, if adopted quickly, is very effective for humans exposed to ricin. In this paper, we aimed to investigate the therapeutic effect of mAb 4C13, a monoclonal antibody against

http://dx.doi.org/10.1016/j.vaccine.2015.06.096 0264-410X/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Dong N, et al. Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.06.096

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ricin [18]. Its antitoxic mechanism was also analyzed based on the results from cell-free system, in vitro and in vivo experiments.

2. Materials and methods 2.1. Ethics statement This study was carried out in strict accordance with the recommendations in the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of National Beijing Center for Drug Safety Evaluation and Research (IACUC 2012-12-002). All efforts were made to minimize suffering.

2.2. Establishment of intoxicated animal model For scanning antitoxic effects of antibodies on ricin poisoning, CD-1 mice (from Vital River Laboratory Animal Technology Co., Ltd, SCXK (Jing) 2012-0001) were intravenously injected (iv) with a lethal dose of ricin (15 ␮g/kg body weight) pre-incubated with saline or monoclonal antibodies (mAbs, 150 ␮g/kg body weight). Ricin was incubated with saline or mAbs at 4 ◦ C for 3 h. To investigate the antitoxic effect of mAb 4C13 as a function of time after ricin poisoning, mAb 4C13 (400 ␮g/kg) was iv injected into mice at different times after administration of 20 ␮g/kg ricin intraperitoneally (ip). The surviving mice were weighed and surviving number of mice was recorded daily. Different doses of mAb 4C13 were used in the therapy of ricin poisoning. Six hours after ip injection with 15 ␮g/kg of ricin, the mice were iv administrated with 0, 25, 100, 200, 400 ␮g/kg of mAb 4C13 and surviving number of mice was recorded. All the mice were provided food and water ad libitum with 12 h dark and light cycle. At the end of experiments the surviving mice were euthanized by narcotizing with CO2 . 2.3. Materials Ricin was obtained from the Laboratory of Toxicant Analysis, Beijing Institute of Pharmacology and Toxicology. In our laboratory the plasimids pET28a RTA (expressed RTA with the same protein sequence encoded by GQ479437.1) and pET28a RTB (expressed RTB with the same protein sequence encoded by JQ617861) were constructed. The recombinant proteins expressed in E. coli were purified by affinity chromatograph. Hybridoma produced mAbs against ricin were prepared by fusing Spleen cells of Balb/C mice immunized with formaldehyde intoxicated ricin [18] and myeloma cells SP2/0 according the standard procedure [19,20]. mAb 4C13, 3D74, 4F2 and 5G6 were purified from mouse ascites (the purity was more than 95% for each) using protein G-sepharose 4 fast flow (Amersham). FITC labeled mAb 4C13, TRITC labeled mAb 3D74 were prepared in our laboratory [21,22]. Caco 2 cells, MDCK cells were from American Type Culture Collection (ATCC). These cell lines were saved in our laboratory. TNT® T3 Coupled Reticulocyte Lysate Systems kits were from Promega (Cat. L4950). HRP-GAM-IgG was from Beijing ZhongShan Golden Bridge Biotechnology Co., Ltd.

2.4. Cell culture Cells were cultured in a MEM/EBSS medium supplemented with 20% of fetal bovine serum, 1% non-essential amino acids, 100 units/ml penicillin and 100 ␮g/ml streptomycin, and incubated at 37 ◦ C in the presence of 5% CO2 . Cells were subcultured when they approximated 80% confluence.

2.5. Cell viability assay First of all, cells were seeded in 96-well cell culture plates at a density of 1 × 105 cells/well. After incubation, the cells were washed with serum-free medium. Ricin and mAb were diluted with serum-free medium. Aliquots of ricin at 80 ng/ml were respectively incubated with the equal volume of 0 ␮g/ml and 1.6 ␮g/ml of antibodies at 37 ◦ C for 1 h and then loaded onto the washed cells. The exposure time points were 24 h and 48 h at 37 ◦ C. At the designated time point, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide (MTT) was added to each well. After 4 h of incubation at 37 ◦ C, 150 ␮l dimethylsulfoxide (DMSO) was added for about 15 min at room temperature after the well content was aspirated to dissolve formazan crystals completely. Absorbance at 490 nm was measured with a micro-ELISA Reader (Varioskan Flash version 2.4.3, Thermo Scientific, America). The optical density of samples without ricin (cells only) was set at 100%. The viability ratio of cells treated with ricin was calculated as: % ratio = (A490sample /A490control ) × 100%.

2.6. The effect of antibody on the inhibition of the protein synthesis induced by ricin TNT® T3 Coupled Reticulocyte Lysate Systems from Promega were used. Both ricin and mAb were diluted with saline. Aliquots of ricin (80 ng/ml) were mixed with an equal volume mAbs (1.6 ␮g/ml) or saline alone and incubated at 4 ◦ C for 1.5 h. 4 ␮l sample was respectively added into the reaction system incubated in ice water to final volume of 20 ␮l. 4 ␮l saline was added to determine the protein synthesis activity as control. After incubation at 30 ◦ C for 1.5 h, the products were cooled in −20 ◦ C for 10 min. 5 ␮l of each reactive product containing synthesized luciferase was mixed with 50 ␮l luciferase assay reagent pre-equilibrated to room temperature and the fluorescence absorbance was measured immediately with the micro-ELISA Reader. 2.7. Binding character of mAb 96-well EIA plates (Costar) were respectively coated with 200 ng/well of RTA, RTB and Ricin in 0.05 M carbonate buffer (pH 9.6) overnight at 4 ◦ C. The plates were washed with PBST (10 mM phosphate-buffered saline containing 0.1% Tween 20, pH 7.4) for three times and blocked with 1.0% bovine serum albumin (BSA) in PBS for 1 h at 37 ◦ C. Antibodies 4F2, 5G6 and 4C13 (1 mg/ml) were diluted with PBST and 100 ␮l diluted sample was added into each well. After 1 h incubation at 37 ◦ C, the plate was washed and 100 ␮l of 1/1000 dilution of HRP-GAM-IgG was added to each well as a second antibody. One hour later, the plate was washed and 100 ␮l TMB substrate solution (10 ml 0.2 mg/ml TMB and 100 ␮l 30% of H2 O2 in 10 ml 0.1 M citrate–0.2 M phosphate buffer, pH 5.2) was added and reacted at room temperature in the dark for 10 min before 50 ␮l of 2 M H2 SO4 was added. The colored reaction product was measured at 450 nm on the micro-ELISA Reader. 2.8. Binding kinetics assays of mAbs to ricin The binding kinetics of mAbs to ricin were measured using Bio-Layer Inter-Ferometry on Octet RED (FartéBio/OctetRED, USA). Sensor tips (AMC) were prewet in 20 mM PBS for 5 min prior to use. The microplate was filled with 200 ␮l of the sample or buffer per well and agitated at 1000 rpm. The mAbs (50 ␮g/ml) were loaded to saturation onto anti-mouse IgG capture biosensors before the loaded biosensors were washed in buffer for 120 s and transferred to wells containing 100, 50 and 25 ␮g/ml of ricin for mAb

Please cite this article in press as: Dong N, et al. Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.06.096

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4F2 or mAb 5G6 and 10, 3 and 1 ␮g/ml of ricin for mAb 4C13. We determined the ricin association and dissociation for 15 min, respectively. All interaction analyses were conducted at 30 ◦ C. The association (kon) and dissociation (koff) rate constants were calculated from a non-linear global fit of the data between the antigen and antibody using the Octet software. Values for the apparent equilibrium dissociation constant (KD ) were calculated from the ratio of koff to kon. 2.9. Fluorescent immunocytochemistry MDCK cells were seeded on glass cover slips sterilized with 75% ethanol in 24-well plates at 5 × 104 cells/well. After growth to about 80% density, cells were cultured in ricin, FITC labeled mAb 4C13, ricin preincubated with FITC labeled mAb 4C13 and ricin preincubated with both of FITC labeled mAb 4C13 and TRITC-labeled mAb 3D74, respectively. DMEM was used to dilute ricin and antibodies. The final concentration at 80 ng/ml of ricin and 1.6 ␮g/ml of antibody were used. The ricin was incubated with DMEM or antibody for 1 h at 37 ◦ C and then loaded onto cells. After intoxication with ricin for 6 h, the supernatant was removed and the slides were rinsed three times with cold PBS. Cells were fixed with cold 95% ethanol for 4 h at 4 ◦ C, washed three times and then blocked for 30 min with 10% goat serum at room temperature. After being permeabilized with 0.1% Triton X-100 in PBS for 1 h and washed three times, cells were incubated with 1 ␮M DAPI (Sigma) to counterstain the cell nuclei. The slips were covered with anti-fade solution (Applygen Technologies Inc., Beijing, China) and detected under a fluorescent microscope (Leica DMI 3000B). The pictures were processed by Image-Pro Plus 6.0.


Table 1 The toxicity of ricin pre-incubated with antibodies in mice. Groups

Mortality of mice

Ricin + saline Ricin + mAb 4F2 Ricin + mAb 5G6 Ricin + mAb 4C13

10/10 10/10 12/12 0/13***

Ricin was incubated with antibodies at 4 ◦ C for 3 h before being iv injected to mice. The dose of ricin was 15 ␮g/kg and that of mAb was 150 ␮g/kg. The mortality rate was observed 10 days after the toxin administration. ***p < 0.001, compared with ricin incubated with saline control. The statistical analysis of the difference of mortality between groups was evaluated by Fisher’ exact of SAS 9.2 statistical analysis software.

immobilized onto the sensor tips following the binding with ricin and dissociation in 20 mM PBS. Kinetic measurements were carried out at three concentrations of ricin respectively (Fig. 3). The result indicated that ricin–mAb 4C13 complex had a lower dissociation rate with KD of 0.165 nM while ricin–mAb 4F2 and ricin–mAb 5G6 had relatively high dissociation rates with KD of 20.0 nM and 10.6 nM. 3.4. The mortality of mice intoxicated with ricin pre-incubated with mAbs

3. Results and discussion

None of the mice intoxicated with 15 ␮g/kg of ricin by iv administration survived in this experiment. The same result was obtained when ricin was pretreated with mAb 4F2 and mAb 5G6. However, the toxicity of ricin in mice was entirely resisted by the pre-treatment with mAb 4C13 (Table 1). This result offered evidence that mAb 4C13 might be of therapeutic value in the treatment of ricin poisoning.

3.1. The effect of mAbs on the cytotoxicity and the protein synthesis inhibitory induced by ricin

3.5. The rescue effect of mAb 4C13 on the mice intoxicated with ricin

Caco 2 and MDCK cells were polarized epithelial cell monolayers that have been used to determine the internalization of ricin and its endocytic mechanism of protein synthesis inhibition [23,24]. In this research, Caco 2 and MDCK cells were used to determine the effect of mAb on the cytotoxicity induced by ricin. When treated with ricin for 24 h and 48 h respectively, the survival rate of Caco 2 cells decreased to 70% and 60%, and that of MDCK decreased to 40% and 30%. The result indicated that only mAb 4C13 had antitoxic activity pretreated with the toxin (Fig. 1A and B). The A chain of ricin specifically modifies 28sRNA by depurination and the depurinated RNA are no longer able to synthesize protein. Our result indicated that protein synthesis was significantly inhibited by ricin and its effect could be significantly diminished by pre-incubation with mAb 4C13 (Fig. 1C and D).

At 2 h, 6 h, 17 h and 24 h time points after ricin intoxication, the mice were iv injected 400 ␮g/kg of mAb 4C13. All mice without mAb treatment died within one week after intoxication. In 30 days, the mortality rate of mice was 0/12 and 3/12 when the mice were treated with mAb 4C13 at 2 h and 6 h time points respectively and no mice died within one week after ricin administration (Fig. 4A). In the repeated experiment, only one in thirteen mice died within one month, indicating that mAb 4C13 could rescue the intoxicated mice although they received 20 ␮g/kg of toxin 6 h before the mAb treatment. The body weight of poisoned mice significantly decreased within one week after intoxication and then increased at the same rate as normal mice (Fig. 4B). Different dosage of mAb 4C13 was administrated 6 h after ricin intoxication. All mice without mAb treatment died within one week after intoxication. When doses above 100 ␮g/kg of mAb were used, more than 80% of the intoxicated mice survived. Although 25 ␮g/kg of mAb 4C13 brought about a survival rate of as low as 6/11, the delayed death time would be still valuable for the therapy of ricin intoxication (Fig. 4C).

3.2. The binding activity of mAb recognized with ricin, RTA and RTB RTA, RTB and ricin were respectively coated onto 96 well plates and incubated with mAb 4F2, mAb 5G6 and mAb 4C13. The mAb binding to the coated antigen was then recognized with HRPGAM-IgG. The result shown in Fig. 2 indicated that the epitopes recognized by mAb 4F2 and mAb 5G6 were respectively located on RTA and RTB with good specificities. Only mAb 4C13 could bind to both of RTA and RTB, presenting better activity with RTA. 3.3. Binding kinetics of mAbs with ricin The affinity constants of mAbs binding with ricin were determined at three concentrations of ricin. Briefly, mAbs were

3.6. The intracellular recognition of the component of mAb 4C13 with ricin MDCK cells were used to trace the cellular internalization of mAb 4C13 combined with ricin. After 6 h incubation with saline and ricin respectively, FITC labeled mAb 4C13 was observed only in the MDCK cells treated with the complex of antigen and antibody (Fig. 5A). But green and red spots in cells were observed after treatment with a sandwich complex of FITC labeled mAb 4C13, ricin and TRITC-labeled mAb 3D74. These antibodies had been used to

Please cite this article in press as: Dong N, et al. Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.06.096

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Survival ratio (%)

Survival ratio (%)

*** P

Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning.

Ricin is a glycoprotein produced in castor seeds and consists of two polypeptide chains named Ricin Toxin A Chain (RTA) and Ricin Toxin B Chain (RTB),...
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