1
Original Article
Isolation of B subunit-specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2
Hideyuki Arimitsu1*, Keiko Sasaki1, Yoshitaka Iba2, Yoshikazu Kurosawa2, Toshiyasu Shimizu1 and Takao Tsuji1
1
Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
2
Innovation Center for Advanced Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan
Running title: Stx2-specific monoclonal antibodies
*Corresponding author Department of Microbiology, Fujita Health University School of Medicine, 1-98, Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan Tel.: +81-562-93-2433; Fax: +81-562-93-4003 E-mail: [email protected]
Subject: Bacteriology (Vaccines)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, Typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1348-0421.12221. This article is protected by copyright. All rights reserved.
2
Abstract Shiga toxin 2 (Stx2)-specific monoclonal antibody (mAb)-producing hybridoma clones were generated from mice. Since mice tend to produce a small amount of B subunit (Stx2B)-specific antibodies at the polyclonal antibody level by immunization through the parenteral route, we immunized Stx2 toxoids intranasally with a mutant heat-labile enterotoxin as a mucosal adjuvant and obtained 11 different hybridoma clones in 2 trials. Among them, 6 were A subunit (Stx2A)-specific and 5 were Stx2B-specific antibody-producing clones. The in vitro neutralization activity of the Stx2B-specific mAbs against Stx2 was higher than that of the Stx2A-specific mAbs on HeLa229 cells. Furthermore, two of the Stx2B-specific mAbs (45 and 75D9) completely inhibited the receptor binding, and also showed in vivo neutralization activity against a 5-fold median lethal dose of Stx2, even at a low concentration in mice. In western blot analysis, these Stx2B-specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the VH and VL regions of clones 45 and 75D9 were determined. Our Stx2B-specific mAbs might be new candidates for the development of mouse-human chimeric Stx2-neutralizing antibodies to reduce the side effects associated with the use of an animal antibody for enterohemorrhagic Escherichia coli infection.
Keywords: monoclonal antibodies, neutralization, Shiga toxins
3
Introduction Enterohemorrhagic Escherichia coli (EHEC; Shiga toxin-producing E. coli) is a food-borne pathogen. Patients with EHEC initially show gastrointestinal symptoms such as abdominal pain, fever, vomiting, and watery diarrhea that then develops into bloody diarrhea, called hemorrhagic colitis. Furthermore, some patients show severe systemic disorders such as hemolytic anemia, thrombocytopenia, and acute renal failure, called hemolytic uremic syndrome (HUS) (1, 2). The major virulence determinants of EHEC are Shiga toxins 1 and 2 (Stx1 and Stx2). Stx consists of an enzymatically active A subunit (Stx1A and Stx2A) and five B subunits (Stx1B and Stx2B), which are responsible for receptor binding (3). Especially, from epidemiological surveys (4-7) and experimental animal data (8, 9), Stx2 demonstrate higher lethality in mice than Stx1, and Stx2-producing strains are considered to be associated with the occurrence of HUS. The administration of antibiotics is controversial for the treatment of EHEC infection because it is associated with the risk of causing severe Shiga toxemia due to toxin-coding prophage induction by some types of drugs (10-13). Therefore, combination therapy with toxin-neutralizing antibodies, in particular ones that inhibit toxin binding to the receptor prior to its incorporation into cells, might be an effective treatment strategy for EHEC infection to decrease the risk of HUS. However, the administration of an animal-derived antibody can induce anaphylaxis. To reduce this risk, the use of human Stx2-specific monoclonal antibodies (mAbs), which are raised in mice that contain transgenes bearing the human Ig loci (14, 15), is likely to be the safest strategy. Alternatively, mouse-human chimeric mAbs are also applicable. Regarding Stx2-specific mAbs, Stx2A-specific (11E10) and Stx2B-specific (VTm1.1) mouse mAbs, both of which neutralized the toxicity of Stx2, have been reported (16, 17). Both antibodies have already been humanized by genetically fusing their variable regions with the human IgG1 constant domain sequences, and tested their neutralization ability (18-20) and pharmacokinetics (21-23) for their clinical application. However, since we consider that the combination of different types of mAb is expected to neutralize toxicity more effectively by binding to other neutralization epitopes simultaneously, we
4
attempted to prepare various types of mAb in this study. The antigenicity of Stx2B, however, tends to be considerably low in mice compared with that observed in rabbits (24) when they are immunized with a genetically attenuated Stx2 toxoid through parenteral routes. Previously, we reported that intranasal immunization of histidine-tagged Stx2B with genetically attenuated mutant heat-labile enterotoxin (mLT) produced by enterotoxigenic E. coli raised Stx2B-specific antibodies in the sera of mice (25, 26). LT and its mutant toxins show powerful adjuvant activity when they are administrated with vaccine antigens through the mucosal route (27). In the present study, we generated Stx2-specific mAb-producing hybridoma clones derived from mice that were immunized intranasally with Stx2 antigens, and characterized their reactivity and toxin-neutralization activity in vitro and in vivo. In addition, we analyzed the amino acid residues on Stx2 that were associated with the neutralization of toxicity by these antibodies.
5
Materials and Methods
Stx2 and mutant Stx2 expression and purification. Stx2 or several different mutant forms of Stx2 (mStx2) were expressed by the E. coli strain MV1184 as reported previously (28, 29). Histidine-tagged Stx2 (Stx2-His) was purified using TALON metal affinity resin (Clontech, Mountain View, CA) and hydroxyapatite chromatography (Bio-Rad, Hercules, CA) (28), and several kinds of Stx2 (without the histidine tag) with a D17N mutation in the B subunit (B:D17N) were purified using an Immobilized D-Galactose Gel (Thermo Fisher Scientific, Rockford, IL) (29). Furthermore, in order to prepare the toxoid, Stx2-His was treated with phosphate-buffered saline (PBS) containing 1% formalin for 3 days at room temperature, followed by dialysis against PBS. Other kinds of mutant Stx2 were prepared from pBSK-Stx2 (29) by site-directed mutagenesis.
Preparation of monoclonal antibodies. In order to obtain hybridoma clones producing Stx2-specific mAbs, we conducted two different experiments using two types of antigen: Stx2-His toxoid in the first trial, while genetically attenuated mStx2 with E167Q and R170L mutations in the A subunit and D17N in the B subunit (A: E167Q+R170L, B: D17N) was used in the second trial to avoid generating anti-histidine tag mAb clones. Five female BALB/c mice (6 weeks of age; Japan SLC, Hamamatsu, Japan) were immunized intranasally 3 times with 15 μl of an immunogen solution containing 10 μg formalin-treated Stx2-His toxoid or mStx2 with 10 μg mLT, in which 3 amino acid residues in the A subunit (R192–I194) were deleted (30), as a mucosal adjuvant, at 2-week intervals. After an additional 2 weeks, each mouse was immunized intraperitoneally with 10 μg of each antigen without adjuvant, and then the spleen was removed at 2 days after immunization. Prepared splenocytes were fused with the mouse myeloma cell line P3U1 by using polyethylene glycol 1500
6
according to the manufacturer’s instructions (Roche Applied Science, Mannheim, Germany). The hybridoma clones were selected with hypoxanthine-aminopterin-thymidine medium containing BM conditioned H1 Hybridoma Cloning Supplement (Roche) for 2 weeks, and then cloned twice by limiting dilution. The hybridoma clones producing Stx2-specific antibodies were selected using an enzyme-linked immunosorbent assay (ELISA). The isotype of each mAb was determined with a Mouse Monoclonal Antibody Isotyping Test Kit (AbD Serotec, Kidlington, UK). In order to obtain highly concentrated mAbs, each hybridoma was injected intraperitoneally into 2 female BALB/c Slc-nu/nu mice (5 weeks of age; Japan SLC), and then ascites were collected at approximately 10 days after injection. The mAbs in the ascites were purified with Protein G-SepharoseTM 4 Fast Flow (GE Healthcare, Uppsala, Sweden) and then dialyzed in PBS.
ELISA. We plated 0.5 μg/50 μl purified Stx2-His onto a 96-well plate (MaxiSorp; Nunc A/S, Roskilde, Denmark) and the plate was incubated overnight at 4°C. After blocking with 100 μl blocking buffer (PBS containing 5% skim milk and 0.05% Tween 20) for 90 min, the plate was reacted with 50 μl of the culture supernatant of the hybridoma or purified antibodies diluted with the blocking buffer for 1 hr, followed by reaction with an alkaline phosphatase-labeled anti-mouse IgG goat antibody (SouthernBiotech, Birmingham, AL) diluted with the blocking buffer for 1 hr. After reaction with 50 µl substrate solution (1 mg/ml p-nitrophenyl phosphate disodium salt hexahydrate, 9.7% diethanolamine, 0.02% NaN3, 0.01% MgCl2·6H2O, pH 9.8) for 30 min, absorbance at 415 nm was measured using an iMarkTM Micro Plate Reader (Bio-Rad). All reaction steps were conducted at 37°C, and each well was washed with 300 μl PBS containing 0.05% Tween 20 (T-PBS) prior to the reactions.
7
Toxin neutralization assay. The in vitro neutralization ability of each mAb was evaluated by a cytotoxicity assay using HeLa229 cells according to the procedure of Neri et al. (31). Two hundred picograms per ml of Stx2 (Nacalai Tesque, Kyoto, Japan) were incubated with an equal volume of each mAb diluted with Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, ampicillin, and streptomycin for 1 hr at 37°C. One hundred microliters of the mixture (final Stx2 concentration was 100 pg/ml, corresponding to an approximately 10-fold median cytotoxic dose [CD50] of the batch used in this study) were added to each well of a 96-well culture plate and then incubated for 72 hr at 37°C. For the in vivo assays, each antibody diluted with PBS was reacted with 35 ng Stx2 (corresponding to a 5-fold median lethal dose [LD50] of the batch used in this study) in a 0.3 ml aliquot for 30 min at 37°C, and was then injected intraperitoneally into at least 5 female ICR mice (4 weeks of age; Japan SLC). The animals were observed for 1 week and their survival was recorded. All animal experiments were carried out in strict accordance with the recommendations of the Regulations for the Management of Laboratory Animals at Fujita Health University. All procedures were approved by the Institutional Animal Care and Use Committee of Fujita Health University (Permit Number: M2301).
Flow cytometry analysis. Seventy-two nanograms of Stx2 (corresponding to approximately 1 pmol), which was preincubated with a 10-fold amount (720 ng) of each mAb for 30 min, was added to 2×105 of HeLa229 cells. After reaction for 30 min, each cell sample was washed with PBS, and then reacted with 1 μg of anti-Stx2-His rabbit IgG which was purified from the antiserum (29) by rProtein A-SepharoseTM Fast Flow (GE Healthcare) for 30 min. After washing, the each cell sample was reacted with 0.5 μg of FITC-labeled anti-rabbit IgG goat F(ab’)2 (Santa Cruz, Dallas, TX) and then analyzed by FACSCaliburTM (Becton Dickinson, Franklin Lakes, NJ). All reaction steps were conducted on ice.
Western blot analysis. Protein samples were separated in a 15% polyacrylamide gel and electroblotted onto a
8
polyvinylidene difluoride (PVDF) membrane. After incubation in a blocking buffer for 1 hr, the membrane was reacted with each mAb or anti-Stx2-His rabbit serum (to detect the loading control reaction with each mStx2 expressed) diluted in PBS containing 5% bovine serum albumin for 1 hr, followed by a reaction with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG goat antibody (Jackson ImmunoResearch, West Grove, PA) or anti-rabbit immunoglobulin swine immunoglobulin (Dako, Copenhagen, Denmark), respectively. Specific bands were detected with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) using an LAS4000 image analyzer (Fujifilm, Tokyo, Japan). All reactions were carried out at room temperature, and the membrane was washed 3 times with T-PBS for 5 min before each reaction.
Determination of the sequences of the VH and VL regions of the mAbs. mRNA from each hybridoma was prepared with Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s method, and then each VH and VL region was amplified independently by RT-PCR using LA-Taq (Takara, Otsu, Japan) and specific primers (listed in Table S1). The amplified products were ligated into the pCR2.1 T-vector (Life Technologies, Carlsbad, CA) and then transformed into TOP10F′ competent cells (Life Technologies). Several colonies, which were picked up and cultured in Luria-Bertani broth containing ampicillin for several hours at 37°C, were used as the template for PCR with a primer set (M13-forward and M13-reverse), and then DNA sequencing was conducted using a Genetic Analyzer Avant3130xl (Applied Biosystems, Foster City, CA). The data were analyzed by Sequencher Ver.4.5 (Gene Codes, Ann Arbor, MI).
Expression of single chain FV-protein A fusion antibodies (scFV-PP) in E. coli. In order to confirm whether the determined sequences were correct, amplified products of each VH and VL gene fragment were ligated into the pSCCA5-E8d phagemid vector, which was modified from pAALSC (32), at the SfiI-NotI sites and NcoI-AscI sites,
9
respectively (as shown in Fig. S1; the nucleotide sequence of pSCCA5-E8d has been submitted to DDBJ under the accession no. AB969670). Furthermore, in order to fuse the double Fc binding domain gene of protein A to the 3′-end of the VL gene, the cp3 gene was eliminated after SalI digestion, and then the resultant plasmid was self-ligated. The E. coli DH12S strain was transformed with each scFV-PP expression plasmid and cultured in 2×YT broth containing ampicillin and 0.1% glucose at 30°C until the exponential growth phase. The expression of scFV-PP was induced by adding isopropyl β-D-1-thiogalactopyranoside at a final concentration of 1 mM and then cultured overnight at 30°C. After centrifugation, each scFV-PP was purified from the mixture of culture supernatant and periplasmic fraction which was extracted from bacterial cells (33) by using IgG-SepharoseTM 6 Fast Flow (GE Healthcare).
Evaluation of the reaction of scFV-PP with Stx2. In order to confirm the validity of the sequences of each mAb, ELISA and western blot analysis were conducted. In ELISA, 0.5 μg/50 μl Stx2 was plated in a 96-well plate and then incubated overnight at 4°C. After incubation with 100 μl blocking buffer for 90 min, the plate was reacted with 50 μl of each scFV-PP or anti-influenza virus-specific Fab-PP (F005-126, as a negative control) (34) diluted with blocking buffer for 1 hr. For reaction with 2×IgG binding domain of protein A (PP) fused at the C-terminal end of the Stx2-specific scFV, each well was reacted with 50 μl HRP-conjugated anti-rabbit immunoglobulin swine immunoglobulin (Dako) diluted with blocking buffer for 1 hr, followed by reaction with 50 μl of the substrate solution for HRP (citrate buffer [pH 5.0] containing 0.04% [wt/vol] o-phenylenediamine and 0.02% [vol/vol] H2O2) for 30 min. After stopping the reaction with 50 μl of 1 M H2SO4, absorbance at 490 nm was measured with a plate reader. In western blot, the cell extract containing Stx2 was blotted onto the PVDF membrane and then blocked in the same manner as described above. The membrane was reacted with each scFV-PP diluted with the blocking buffer, followed by reaction with HRP-conjugated anti-sheep IgG (H&L) rabbit antibody (Rockland Immunochemicals, Limerick, PA) through the interaction with PP of scFV.
10
Primer sequences. All primer sequences used in this study are listed in our previous papers (28, 29) and in Table S1 of this manuscript.
11
Results
Isolation of Stx2-specific mAb clones. In this study, we screened 4 clones that produced Stx2-His-specific antibodies using ELISA in the first trial (Fig. 1a). Since each mAb was screened with Stx2-His, we attempted to confirm the specificity of each mAb against non-tagged Stx2 in cell extracts by western blot analysis to exclude the possibility of a histidine-tag-specific reaction. mAbs 40 and 131 reacted only to Stx2A, whereas mAb 45 reacted only to Stx2B, indicating that clones 40 and 131 were Stx2A-specific mAb-producing clones, while clone 45 was an Stx2B-specific mAb-producing clone. Conversely, mAb 134 did not react with either the A or B bands in western blot analysis. In addition, since mAb 134 reacted to another histidine-tagged protein that we had prepared previously (data not shown), we concluded that mAb 134 was a histidine tag-specific antibody that was applicable as the negative control in this study. In the second trial, since we had previously developed an affinity purification method for the mStx2 antigen without the use of any tag proteins (29), we used an improved mStx2 (A:E167Q+R170L, B:D17N) as an antigen to avoid generating histidine tag antibody-producing clones, and obtained 4 Stx2A-specific (clones 59A1, 64E5, 68G11, and 70F5) and 4 Stx2B-specific (clones 61C6, 72F2, 73B4, and 75D9) mAb-producing clones (Fig. 1b). All antibodies obtained in this study were of the IgG1 heavy chain and kappa light chain classes.
In vitro neutralization effects of each mAb against Stx2. In order to analyze the neutralization activity of each mAb against Stx2, in vitro neutralization assays were conducted by using HeLa229 cells. For the 4 antibodies obtained in the first trial, Stx2B-specific mAb 45 showed the highest neutralization activity against Stx2 (Fig. 2a). Stx2A-specific mAb 131 also showed a higher neutralization effect than the other Stx2A-specific mAb (mAb 40) and the negative control (mAb 134),
12
although it was considerably weaker than mAb 45. Similarly, among the 8 antibodies obtained in the second trial, 4 different Stx2B-specific antibodies (61C6, 72F2, 73B4, and 75D9) showed a stronger neutralization effect against Stx2 than the 4 different Stx2A-specific antibodies (59A1, 64E5, 68G11, and 70F5; Fig. 2b); especially, mAb 75D9 showed the highest neutralization activity among them. Although some Stx2A-specific mAbs showed neutralization activity, their activity was lower than that of mAbs 45 and 75D9 in each trial. Therefore, we focused on the two mAb clones 45 and 75D9 and analyzed their in vivo neutralization activity.
Inhibitory effect of mAbs for Stx2 binding to HeLa229 cells. Since we hypothesized that higher in vitro neutralization activities of Stx2B-specific mAbs 45 and 75D9 than that of Stx2A-specific mAbs were related to inhibition of receptor binding, the binding inhibition of each mAb against Stx2 to HeLa229 cells was analyzed using flow cytometry analysis. As shown in Fig. 3, a ten-fold amount of Stx2B-specific neutralizing mAbs 45 and 75D9 completely inhibited the binding of 72 ng of Stx2, whereas that of negative control mAb 134 did not show any inhibition of Stx2 binding. Regarding the Stx2A-specific mAbs, although two kinds of neutralizing mAbs 131 and 70F5 also partially inhibited Stx2 binding and non-neutralizing mAb 64E5 did not, these results are not reproducible in some trials (data not shown). These results indicate at least that high neutralization activities of mAbs 45 and 75D9 against Stx2 are based on the complete inhibition of the receptor binding.
In vivo neutralization effects of mAbs against Stx2. Each Stx2A- and Stx2B-specific mAb which showed the highest in vitro neutralization activity in each trial was administered intraperitoneally to mice with Stx2. As shown in Table 1, 1000 ng of each Stx2B-specific mAbs (45 and 75D9) completely protected the mice from a 5-fold LD50 of Stx2, whereas that of each Stx2A-specific mAbs (131 and 70F5) partially protected. Especially, in the case of 75D9, a complete protection
13
effect was retained at 100 ng. mAb 134 (as a negative control) did not show any neutralization activity, even at 1000 ng. From the dose-dependent protection afforded by these antibodies, mAb 75D9 seemed to be the most effective antibody prepared in this study, leading to the correlation with the in vitro assays.
Identification of the amino acid residues of Stx2 associated with recognition by each mAb. In vitro and in vivo neutralization assays revealed that mAbs 45 and 75D9 are alternative candidates for the development of mouse-human chimeric Stx2-neutralizing antibodies. Therefore, in order to identify the amino acid residues in Stx2 that were recognized by mAbs 45 and 75D9, western blot analysis was conducted against the prepared mStx2 clones. The locations of the mutations introduced to the B subunit were selected to focus on the receptor binding sites (W29, S54, E57) (16, 35) and the different residues (K5, D16, D17, D24, K26, S31, K52, S54, E57, E64, Q66, N69, D70) from those in the other variant toxins (Stx2c, Stx2d, Stx2e, and Stx2f). As shown in Fig. 4a, both mAbs that neutralized the toxicity of Stx2 did not react to the single W29A, S54N, and E57S mutants, each amino acid residue of which is associated with receptor binding (16, 35). These results indicate that the Stx2B-specific mAbs 45 and 75D9 neutralize the toxicity of Stx2 by binding to the receptor binding sites of the B subunit as shown in Fig. 4b.
Amino acid sequences of the VH and VL regions of mAbs 45 and 75D9. Additionally, we compared the amino acid sequences of the VH and VL regions of mAbs 45 and 75D9 deduced from their nucleotide sequences. The sequences of the VH and VL regions have been submitted to the DDBJ under the accession nos. AB969666, AB969667, AB969668, and AB969669 (as Stx2-45-VH, Stx2-45-VL, Stx2-75-VH, and Stx2-75-VL, respectively). The validity of the sequences of each mAb was confirmed by the expression of the scFV-PP of each clone (scFV-PP 45, scFV-PP 75D9) in the E. coli DH12S strain. As shown in Fig. 5, we confirmed the reactivity of each scFV-PP against the Stx2B both in ELISA and
14
western blot analysis. The amino acid sequences of the variable region between both antibodies were quite different, indicating that each antibody was derived from a different clone.
15
Discussion In this study, we obtained 11 different Stx2-specific mAb-producing hybridomas following the intranasal immunization of mice with Stx2 antigens and mLT; 6 were Stx2A-specific and 5 were Stx2B-specific mAb-producing clones. Although mice tend to produce a small amount of Stx2B-specific antibody at the polyclonal antibody level (24), we reported previously that intranasal immunization of histidine-tagged Stx2B with mLT induced the production of Stx2B-specific antibodies that neutralized the toxicity of Stx2 in the sera of mice (25, 26). Although the reason why mLT induces different immunoreactivities against Stx2 by intranasal immunization is unknown, these results indicate that mucosal immunization with mLT might be a useful strategy to raise antibodies against antigens that cannot induce specific antibodies by parenteral (subcutaneous or intramuscular) routes. Although our ultimate purpose was to obtain the Stx2-specific mAbs which cross-reacted with Stx1, we confirmed that none of the mAbs cross-reacted with Stx1 in the early process of screening Stx2-specific mAb hybridoma clones (data not shown). In the neutralization assay using HeLa229 cells, the Stx2B-specific antibodies obtained in this study tended to show a higher neutralization effect than the Stx2A-specific mAbs against Stx2, and the highest neutralizing mAb clones (45 and 75D9) prepared in each trial showed strong protective effects on mice against a 5-fold LD50 Stx2 challenge. Nakao et al. reported that 50 % neutralizing concentration of anti-Stx2B mAb VTm1.1 against 125 pg/ml of Stx2 on ACHN cells was approximately 55 ng/ml (16). On the other hand, Edwards et al. reported that the amount of mAb 11E10 required to neutralize 1 CD50 of Stx2 (corresponding to 1 pg used in their study) on Vero cells was calculated at 2.7 ng (18). In the case of our mAb, 50 % neutralizing concentration of the mAb 75D9 against 100 pg/ml of Stx2 on HeLa229 cells was between 1.6-8 ng/ml (Fig. 2). These indicate that 75D9 has especially strong neutralization activity which is comparable to VTm 1.1 and 11E10, although the assay methods are different from each other.
16
The flow cytometry analysis using HeLa229 cells revealed that the mAbs 45 and 75D9 directly inhibited the binding of Stx2 to the receptor. Furthermore, neither Stx2B-specific mAbs reacted to 3 different B subunit mutants (W29A, S54N, and E57S). It was reported that VTm1.1 neutralized the toxicity of Stx2 by binding to amino acid E56 (corresponding to E57 in our study) in Stx2B (16). Since the crystal structure of Stx2 had not been reported at that time, they speculated upon the location of E57 in the B subunit by comparing it to the corresponding amino acid of Stx1, and suggested that E57 was associated with receptor binding (16). According to the crystal structure of Stx2 reported by Fraser et al. (35), E57 is located near W29, which is associated with receptor binding (Fig. 4b). Furthermore, they mentioned the association of S54 with receptor binding (35). These indicate that mAbs 45 and 75D9 neutralize the toxicity of Stx2 by binding to the receptor binding site of the B subunit. On the other hand, Stx2A-specific mAbs 131 and 70F5, which partially neutralized the toxicities of Stx2, partially inhibited the binding of Stx2 to HeLa229 cells. However, since these inhibitions were not reproducible in some trials, we could not discuss whether or not these Stx2A-specific mAbs partially neutralized the toxicity through physical inhibition of the receptor binding. As shown in Fig. 2, the neutralization activity of the remaining 3 Stx2B-specific mAbs (61C6, 72F2, and 73B4) prepared in the second trial was also at the same level as that of mAb 45 prepared in the first trial, and their reaction profiles against mStx2s in western blot analysis were also similar to mAbs 45 and 75D9 (data not shown). These results indicate that these antibodies neutralize toxicity by inhibiting receptor binding, and that the neutralization epitopes on the B subunit of Stx2 are limited to a specific region associated with receptor binding, as shown in Fig. 4b. In this study, we determined the nucleotide sequences of the VH and VL regions of mAbs 45 and 75D9 for the development of mouse-human chimeric Stx2-neutralizing antibodies. We confirmed the validity of the sequences of each mAb by Stx2-specific ELISA and western blot using each scFV-PP protein prepared. The reactivity of scFV-PP of each clone against Stx2 on western blot was lower than that of original mAb, due to the fact that the scFV is a monovalent
17
antibody. Additionally, since the principle of each method used in scFv-PP is somewhat different from that used in the original mAb from the point of using interaction of scFV-PP with secondary antibodies through the Fc region, we can only evaluate the validity of the each sequences in specificities, but not in sensitivities compared with the original mAb. We still have several different candidate mAbs that neutralize the toxicity of Stx2, including Stx2A-specific neutralization mAbs (61C6, 68G11, 72F2, 73B4). Even in the case that each mAb recognizes similar epitopes, their neutralization effect against the toxin is also dependent on their affinity to the neutralization epitopes. Although mAbs 45 or 75D9 show a sufficient neutralization effect even in independently conducted trials, we need to investigate the combination effects of our mAbs on effective Stx2-neutralization in the near future.
18
Acknowledgements
This work was supported, in part, by JSPS KAKENHI (Number 24590539), the Strategic Research Base Development Program for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and Health and Labor Sciences Research Grants for Research on Global Health Issues from the Ministry of Health, Labor, and Welfare, Japan.
Disclosure The authors declare no conflicts of interest or financial support.
19
References
1
Tarr P.I., Gordon C.A.,Chandler W.L. (2005) Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet
2
365: 1073-86.
Karmali M.A. (2004) Infection by Shiga toxin-producing Escherichia coli: an overview. Mol Biotechnol 26: 117-22.
3
O'Brien A.D., Tesh V.L., Donohue-Rolfe A., Jackson M.P., Olsnes S., Sandvig K., Lindberg A.A.,Keusch G.T. (1992) Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis. Curr Top Microbiol Immunol
4
180: 65-94.
Boerlin P., McEwen S.A., Boerlin-Petzold F., Wilson J.B., Johnson R.P.,Gyles C.L. (1999) Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. J Clin Microbiol
37:
497-503. 5
Ostroff S.M., Tarr P.I., Neill M.A., Lewis J.H., Hargrett-Bean N.,Kobayashi J.M. (1989) Toxin genotypes and plasmid profiles as determinants of systemic sequelae in Escherichia coli O157:H7 infections. J Infect Dis
160:
994-8. 6
Kleanthous H., Smith H.R., Scotland S.M., Gross R.J., Rowe B., Taylor C.M.,Milford D.V. (1990) Haemolytic uraemic syndromes in the British Isles, 1985-8: association with verocytotoxin producing Escherichia coli. Part 2: Microbiological aspects. Arch Dis Child
7
65: 722-7.
Scotland S.M., Willshaw G.A., Smith H.R.,Rowe B. (1987) Properties of strains of Escherichia coli belonging to serogroup O157 with special reference to production of Vero cytotoxins VT1 and VT2. Epidemiol Infect
99:
613-24. 8
Takeda Y., Kurazono H.,Yamasaki S. (1993) Vero toxins (Shiga-like toxins) produced by enterohemorrhagic Escherichia coli (verocytotoxin-producing E. coli). Microbiol Immunol
37: 591-9.
20
9
Tesh V.L., Burris J.A., Owens J.W., Gordon V.M., Wadolkowski E.A., O'Brien A.D.,Samuel J.E. (1993) Comparison of the relative toxicities of Shiga-like toxins type I and type II for mice. Infect Immun
61:
3392-402. 10
Zhang X., McDaniel A.D., Wolf L.E., Keusch G.T., Waldor M.K.,Acheson D.W. (2000) Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. J Infect Dis
11
Matsushiro A., Sato K., Miyamoto H., Yamamura T.,Honda T. (1999) Induction of prophages of enterohemorrhagic Escherichia coli O157:H7 with norfloxacin. J Bacteriol
12
181: 2257-60.
Walterspiel J.N., Ashkenazi S., Morrow A.L.,Cleary T.G. (1992) Effect of subinhibitory concentrations of antibiotics on extracellular Shiga-like toxin I. Infection
13
20: 25-9.
Wong C.S., Jelacic S., Habeeb R.L., Watkins S.L.,Tarr P.I. (2000) The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N Engl J Med
14
181: 664-70.
342: 1930-6.
Mukherjee J., Chios K., Fishwild D., Hudson D., O'Donnell S., Rich S.M., Donohue-Rolfe A.,Tzipori S. (2002) Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection. Infect Immun
15
70: 612-9.
Sheoran A.S., Chapman S., Singh P., Donohue-Rolfe A.,Tzipori S. (2003) Stx2-specific human monoclonal antibodies protect mice against lethal infection with Escherichia coli expressing Stx2 variants. Infect Immun 71: 3125-30.
16
Nakao H., Kiyokawa N., Fujimoto J., Yamasaki S.,Takeda T. (1999) Monoclonal antibody to Shiga toxin 2 which blocks receptor binding and neutralizes cytotoxicity. Infect Immun
17
67: 5717-22.
Perera L.P., Marques L.R.,O'Brien A.D. (1988) Isolation and characterization of monoclonal antibodies to Shiga-like toxin II of enterohemorrhagic Escherichia coli and use of the monoclonal antibodies in a colony enzyme-linked immunosorbent assay. J Clin Microbiol
18
26: 2127-31.
Edwards AC M.-C.A., Arbuthnott K, Stinson JR, Schmitt CK, Wong HC, O'Brien A.D. (1998) Vero cell neutralization and mouse protective efficacy of humanized monoclonal antibodies against Escherichia coli toxins
21
Stx1 and Stx2, p 388–392. In Kaper JB, O'Brien AD, editors (ed), Escherichia coli O157:H7 and other Shiga toxin-producing E coli strains ASM Press, Washington, DC. 19
Kimura T., Co M.S., Vasquez M., Wei S., Xu H., Tani S., Sakai Y., Kawamura T., Matsumoto Y., Nakao H.,Takeda T. (2002) Development of humanized monoclonal antibody TMA-15 which neutralizes Shiga toxin 2. Hybrid Hybridomics
20
21: 161-8.
Yamagami S., Motoki M., Kimura T., Izumi H., Takeda T., Katsuura Y.,Matsumoto Y. (2001) Efficacy of postinfection treatment with anti-Shiga toxin (Stx) 2 humanized monoclonal antibody TMA-15 in mice lethally challenged with Stx-producing Escherichia coli. J Infect Dis
21
184: 738-42.
Bitzan M., Poole R., Mehran M., Sicard E., Brockus C., Thuning-Roberson C.,Rivière M. (2009) Safety and pharmacokinetics of chimeric anti-Shiga toxin 1 and anti-Shiga toxin 2 monoclonal antibodies in healthy volunteers. Antimicrob Agents Chemother
22
53: 3081-7.
Dowling T.C., Chavaillaz P.A., Young D.G., Melton-Celsa A., O'Brien A., Thuning-Roberson C., Edelman R.,Tacket C.O. (2005) Phase 1 safety and pharmacokinetic study of chimeric murine-human monoclonal antibody cαStx2 administered intravenously to healthy adult volunteers. Antimicrob Agents Chemother
23
49: 1808-12.
López E.L., Contrini M.M., Glatstein E., González Ayala S., Santoro R., Allende D., Ezcurra G., Teplitz E., Koyama T., Matsumoto Y., Sato H., Sakai K., Hoshide S., Komoriya K., Morita T., Harning R.,Brookman S. (2010) Safety and pharmacokinetics of urtoxazumab, a humanized monoclonal antibody, against Shiga-like toxin 2 in healthy adults and in pediatric patients infected with Shiga-like toxin-producing Escherichia coli. Antimicrob Agents Chemother
24
54: 239-43.
Wen S., Teel L., Judge N.,O'Brien A.D. (2006) Genetic toxoids of Shiga toxin types 1 and 2 protect mice against homologous but not heterologous toxin challenge. Vaccine
25
24: 1142-48.
Tsuji T., Shimizu T., Sasaki K., Shimizu Y., Tsukamoto K., Arimitsu H., Ochi S., Sugiyama S., Taniguchi K., Neri P.,Mori H. (2008) Protection of mice from Shiga toxin-2 toxemia by mucosal vaccine of Shiga toxin 2B-His with Escherichia coli enterotoxin. Vaccine
26
26: 469-76.
Tsuji T., Shimizu T., Sasaki K., Tsukamoto K., Arimitsu H., Ochi S., Taniguchi K., Noda M., Neri P.,Mori H.
22
(2008) A nasal vaccine comprising B-subunit derivative of Shiga toxin 2 for cross-protection against Shiga toxin types 1 and 2. Vaccine 27
26: 2092-9.
Connell T.D. (2007) Cholera toxin, LT-I, LT-IIa and LT-IIb: the critical role of ganglioside binding in immunomodulation by type I and type II heat-labile enterotoxins. Expert Rev Vaccines
28
Arimitsu H., Sasaki K., Shimizu T., Tsukamoto K., Shimizu T.,Tsuji T. (2013) Large-scale preparation of Shiga toxin 2 in Escherichia coli for toxoid vaccine antigen production. Microbiol Immunol
29
57: 38-45.
Arimitsu H., Sasaki K., Kojima H., Yanaka T.,Tsuji T. (2013) Simple Method for Shiga Toxin 2e Purification by Affinity Chromatography via Binding to the Divinyl Sulfone Group. PLoS One
30
6: 821-34.
8: e83577.
Tsuji T., Yokochi T., Kamiya H., Kawamoto Y., Miyama A.,Asano Y. (1997) Relationship between a low toxicity of the mutant A subunit of enterotoxigenic Escherichia coli enterotoxin and its strong adjuvant action. Immunology
31
90: 176-82.
Neri P., Nagano S.I., Yokoyama S., Dohi H., Kobayashi K., Miura T., Inazu T., Sugiyama T., Nishida Y.,Mori H. (2007) Neutralizing activity of polyvalent Gb3, Gb2 and galacto-trehalose models against Shiga toxins. Microbiol Immunol
32
51: 581-92.
Iba Y., Ito W.,Kurosawa Y. (1997) Expression vectors for the introduction of highly diverged sequences into the six complementarity-determining regions of an antibody. Gene
33
Skerra A.,Plückthun A. (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science
34
194: 35-46.
240: 1038-41.
Iba Y., Fujii Y., Ohshima N., Sumida T., Kubota-Koketsu R., Ikeda M., Wakiyama M., Shirouzu M., Okada J., Okuno Y., Kurosawa Y.,Yokoyama S. (2014) Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses. J Virol
35
88: 7130-44.
Fraser M.E., Fujinaga M., Cherney M.M., Melton-Celsa A.R., Twiddy E.M., O'Brien A.D.,James M.N. (2004) Structure of Shiga Toxin Type 2 (Stx2) from Escherichia coli O157:H7. J Biol Chem
279: 27511-7.
23
Figure Legends
Fig. 1. Immunological properties of the mAbs.
Antibody level of mAbs obtained by immunizing Stx2-His toxoid in
the first trial (a) and by immunizing mStx2 in the second trial (b). Stx2-His was used as an immobilized antigen of ELISA in each trial. The data represent the mean of three separate experiments and the error bars indicate SEM. The data of the B subunit-specific antibodies are represented as closed color symbols. The western blot analysis of each mAb to 5 μl of cell extract from non-tagged Stx2-expressing E. coli MV1184 is shown on the right panel of each trial. Each lane was separated after blocking, and combined again in the detection step. The concentration of each mAb in western blot analysis was as follows:
40 (1 μg/ml); 45 (3 μg/ml); 131 (2.5 μg/ml); 134 (1 μg/ml); 59A1 (10 ng/ml); 61C6 (1 μg/ml);
64E5 (5 ng/ml); 68G11 (500 ng/ml); 70F5 (2.5 μg/ml); 72F2 (1 μg/ml); 73B4 (1 μg/ml); 75D9 (1 μg/ml).
Fig. 2. In vitro neutralization activity of each mAb against Stx2. In the first (a) and second (b) trials. The data represent the mean of three separate experiments and the error bars indicate SEM. The data of the B subunit-specific antibodies are represented as closed color symbols.
Fig. 3. Inhibitory effect of mAbs against Stx2 binding to HeLa229 cells. Stx2 (72 ng) was preincubated with 10-fold amount (720ng) of each mAb, and then added to 2×105 of the cells. Binding level of toxins on the cell surface was detected by anti-Stx2-His rabbit IgG, followed by FITC-labeled anti-rabbit IgG goat F(ab’) 2. Open black histgrams; untreated cells, shaded histgrams; Stx2-treated cells, open colored histgrams; Stx2-mAb mixture-treated cells.
24
Fig. 4. Identification of the amino acid residues of Stx2B associated with the binding of mAbs 45 and 75D9. (a) Western blot analysis of the mAbs 45 (3 μg/ml) and 75D9 (1 μg/ml) against 5 μl cell extract containing each mStx2 with a mutation in the B subunit. Loading control reactions against each mStx2 were detected by anti-Stx2-His rabbit serum (polyclonal). The mutated positions are shown at the bottom of the panel. (b) Location of the amino acid residues on the B subunit pentamer associated with the binding of mAbs 45 and 75D9. Diagonal bottom view of the crystal structure of Stx2 modified from PDB ID 1R4P, reported by Fraser et al. (35). The residues assessed are shown in blue (W29), green (S54), and red (E57). The A subunit is shown in gray.
Fig. 5. Reactivities of the scFV-PP 45 and scFV-PP 75D9 against Stx2. a) Each diluted scFV-PP, which was purified from DH12S transformed with the scFV-PP expression plasmid of each Stx2B-specific mAb clone, was reacted with 0.5 μg/50 μl of Stx2, followed by reaction with HRP-conjugated anti-rabbit immunoglobulin swine immunoglobulin (Dako). Purified influenza virus-specific Fab-PP was used as a negative control in this assay. b) The western blot analysis of each scFV-PP to the cell extract from non-tagged Stx2-expressing E. coli MV1184.
Fig. S1. Schematic diagram of the single chain gene cloning site of the scFV-PP expression plasmid pSCCA5-E8d.
Table S1. Oligonucleotide primers used in this study.
25
List of Abbreviations
CD50, median cytotoxic dose EHEC, Enterohemorrhagic Escherichia coli ELISA, enzyme-linked immunosorbent assay HRP, horseradish peroxidase HUS, hemolytic uremic syndrome LD50, median lethal dose mAb, monoclonal antibody mLT, mutant heat-labile enterotoxin mStx2, mutant Shiga toxin 2 PBS, phosphate-buffered saline Stx2, Shiga toxin 2 Stx2A, Shiga toxin 2 A subunit Stx2B, Shiga toxin 2 B subunit
26
Table 1. In vivo neutralization activity of Stx2-specific mAbs against Stx2†‡ Amonut of mAb (ng) mixed with 35ng Stx2 Clone No. 1,000
100
50
30
10
45
5/5
3/5
2/5
ND
0/5
75D9
5/5
5/5
ND
3/5
1/5
131
2/5
1/5
ND
ND
ND
70F5
2/5
0/5
ND
ND
ND
134
0/5
ND
ND
ND
ND
†
35 ng (corresponds to 5-fold LD50) of Stx2 were inoculated intraperitoneally with each diluted mAb.
‡
Result is shown as the number of survived / challenged mice.
ND: Not determined
27
Figure 1
28
Figure 2
29
Figure 3
30
Figure 4
31
Figure 5
Original Article
Isolation of B subunit-specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2
Hideyuki Arimitsu1*, Keiko Sasaki1, Yoshitaka Iba2, Yoshikazu Kurosawa2, Toshiyasu Shimizu1 and Takao Tsuji1
1
Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
2
Innovation Center for Advanced Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan
Running title: Stx2-specific monoclonal antibodies
*Corresponding author Department of Microbiology, Fujita Health University School of Medicine, 1-98, Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan Tel.: +81-562-93-2433; Fax: +81-562-93-4003 E-mail: [email protected]
Subject: Bacteriology (Vaccines)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, Typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1348-0421.12221. This article is protected by copyright. All rights reserved.
2
Abstract Shiga toxin 2 (Stx2)-specific monoclonal antibody (mAb)-producing hybridoma clones were generated from mice. Since mice tend to produce a small amount of B subunit (Stx2B)-specific antibodies at the polyclonal antibody level by immunization through the parenteral route, we immunized Stx2 toxoids intranasally with a mutant heat-labile enterotoxin as a mucosal adjuvant and obtained 11 different hybridoma clones in 2 trials. Among them, 6 were A subunit (Stx2A)-specific and 5 were Stx2B-specific antibody-producing clones. The in vitro neutralization activity of the Stx2B-specific mAbs against Stx2 was higher than that of the Stx2A-specific mAbs on HeLa229 cells. Furthermore, two of the Stx2B-specific mAbs (45 and 75D9) completely inhibited the receptor binding, and also showed in vivo neutralization activity against a 5-fold median lethal dose of Stx2, even at a low concentration in mice. In western blot analysis, these Stx2B-specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the VH and VL regions of clones 45 and 75D9 were determined. Our Stx2B-specific mAbs might be new candidates for the development of mouse-human chimeric Stx2-neutralizing antibodies to reduce the side effects associated with the use of an animal antibody for enterohemorrhagic Escherichia coli infection.
Keywords: monoclonal antibodies, neutralization, Shiga toxins
3
Introduction Enterohemorrhagic Escherichia coli (EHEC; Shiga toxin-producing E. coli) is a food-borne pathogen. Patients with EHEC initially show gastrointestinal symptoms such as abdominal pain, fever, vomiting, and watery diarrhea that then develops into bloody diarrhea, called hemorrhagic colitis. Furthermore, some patients show severe systemic disorders such as hemolytic anemia, thrombocytopenia, and acute renal failure, called hemolytic uremic syndrome (HUS) (1, 2). The major virulence determinants of EHEC are Shiga toxins 1 and 2 (Stx1 and Stx2). Stx consists of an enzymatically active A subunit (Stx1A and Stx2A) and five B subunits (Stx1B and Stx2B), which are responsible for receptor binding (3). Especially, from epidemiological surveys (4-7) and experimental animal data (8, 9), Stx2 demonstrate higher lethality in mice than Stx1, and Stx2-producing strains are considered to be associated with the occurrence of HUS. The administration of antibiotics is controversial for the treatment of EHEC infection because it is associated with the risk of causing severe Shiga toxemia due to toxin-coding prophage induction by some types of drugs (10-13). Therefore, combination therapy with toxin-neutralizing antibodies, in particular ones that inhibit toxin binding to the receptor prior to its incorporation into cells, might be an effective treatment strategy for EHEC infection to decrease the risk of HUS. However, the administration of an animal-derived antibody can induce anaphylaxis. To reduce this risk, the use of human Stx2-specific monoclonal antibodies (mAbs), which are raised in mice that contain transgenes bearing the human Ig loci (14, 15), is likely to be the safest strategy. Alternatively, mouse-human chimeric mAbs are also applicable. Regarding Stx2-specific mAbs, Stx2A-specific (11E10) and Stx2B-specific (VTm1.1) mouse mAbs, both of which neutralized the toxicity of Stx2, have been reported (16, 17). Both antibodies have already been humanized by genetically fusing their variable regions with the human IgG1 constant domain sequences, and tested their neutralization ability (18-20) and pharmacokinetics (21-23) for their clinical application. However, since we consider that the combination of different types of mAb is expected to neutralize toxicity more effectively by binding to other neutralization epitopes simultaneously, we
4
attempted to prepare various types of mAb in this study. The antigenicity of Stx2B, however, tends to be considerably low in mice compared with that observed in rabbits (24) when they are immunized with a genetically attenuated Stx2 toxoid through parenteral routes. Previously, we reported that intranasal immunization of histidine-tagged Stx2B with genetically attenuated mutant heat-labile enterotoxin (mLT) produced by enterotoxigenic E. coli raised Stx2B-specific antibodies in the sera of mice (25, 26). LT and its mutant toxins show powerful adjuvant activity when they are administrated with vaccine antigens through the mucosal route (27). In the present study, we generated Stx2-specific mAb-producing hybridoma clones derived from mice that were immunized intranasally with Stx2 antigens, and characterized their reactivity and toxin-neutralization activity in vitro and in vivo. In addition, we analyzed the amino acid residues on Stx2 that were associated with the neutralization of toxicity by these antibodies.
5
Materials and Methods
Stx2 and mutant Stx2 expression and purification. Stx2 or several different mutant forms of Stx2 (mStx2) were expressed by the E. coli strain MV1184 as reported previously (28, 29). Histidine-tagged Stx2 (Stx2-His) was purified using TALON metal affinity resin (Clontech, Mountain View, CA) and hydroxyapatite chromatography (Bio-Rad, Hercules, CA) (28), and several kinds of Stx2 (without the histidine tag) with a D17N mutation in the B subunit (B:D17N) were purified using an Immobilized D-Galactose Gel (Thermo Fisher Scientific, Rockford, IL) (29). Furthermore, in order to prepare the toxoid, Stx2-His was treated with phosphate-buffered saline (PBS) containing 1% formalin for 3 days at room temperature, followed by dialysis against PBS. Other kinds of mutant Stx2 were prepared from pBSK-Stx2 (29) by site-directed mutagenesis.
Preparation of monoclonal antibodies. In order to obtain hybridoma clones producing Stx2-specific mAbs, we conducted two different experiments using two types of antigen: Stx2-His toxoid in the first trial, while genetically attenuated mStx2 with E167Q and R170L mutations in the A subunit and D17N in the B subunit (A: E167Q+R170L, B: D17N) was used in the second trial to avoid generating anti-histidine tag mAb clones. Five female BALB/c mice (6 weeks of age; Japan SLC, Hamamatsu, Japan) were immunized intranasally 3 times with 15 μl of an immunogen solution containing 10 μg formalin-treated Stx2-His toxoid or mStx2 with 10 μg mLT, in which 3 amino acid residues in the A subunit (R192–I194) were deleted (30), as a mucosal adjuvant, at 2-week intervals. After an additional 2 weeks, each mouse was immunized intraperitoneally with 10 μg of each antigen without adjuvant, and then the spleen was removed at 2 days after immunization. Prepared splenocytes were fused with the mouse myeloma cell line P3U1 by using polyethylene glycol 1500
6
according to the manufacturer’s instructions (Roche Applied Science, Mannheim, Germany). The hybridoma clones were selected with hypoxanthine-aminopterin-thymidine medium containing BM conditioned H1 Hybridoma Cloning Supplement (Roche) for 2 weeks, and then cloned twice by limiting dilution. The hybridoma clones producing Stx2-specific antibodies were selected using an enzyme-linked immunosorbent assay (ELISA). The isotype of each mAb was determined with a Mouse Monoclonal Antibody Isotyping Test Kit (AbD Serotec, Kidlington, UK). In order to obtain highly concentrated mAbs, each hybridoma was injected intraperitoneally into 2 female BALB/c Slc-nu/nu mice (5 weeks of age; Japan SLC), and then ascites were collected at approximately 10 days after injection. The mAbs in the ascites were purified with Protein G-SepharoseTM 4 Fast Flow (GE Healthcare, Uppsala, Sweden) and then dialyzed in PBS.
ELISA. We plated 0.5 μg/50 μl purified Stx2-His onto a 96-well plate (MaxiSorp; Nunc A/S, Roskilde, Denmark) and the plate was incubated overnight at 4°C. After blocking with 100 μl blocking buffer (PBS containing 5% skim milk and 0.05% Tween 20) for 90 min, the plate was reacted with 50 μl of the culture supernatant of the hybridoma or purified antibodies diluted with the blocking buffer for 1 hr, followed by reaction with an alkaline phosphatase-labeled anti-mouse IgG goat antibody (SouthernBiotech, Birmingham, AL) diluted with the blocking buffer for 1 hr. After reaction with 50 µl substrate solution (1 mg/ml p-nitrophenyl phosphate disodium salt hexahydrate, 9.7% diethanolamine, 0.02% NaN3, 0.01% MgCl2·6H2O, pH 9.8) for 30 min, absorbance at 415 nm was measured using an iMarkTM Micro Plate Reader (Bio-Rad). All reaction steps were conducted at 37°C, and each well was washed with 300 μl PBS containing 0.05% Tween 20 (T-PBS) prior to the reactions.
7
Toxin neutralization assay. The in vitro neutralization ability of each mAb was evaluated by a cytotoxicity assay using HeLa229 cells according to the procedure of Neri et al. (31). Two hundred picograms per ml of Stx2 (Nacalai Tesque, Kyoto, Japan) were incubated with an equal volume of each mAb diluted with Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, ampicillin, and streptomycin for 1 hr at 37°C. One hundred microliters of the mixture (final Stx2 concentration was 100 pg/ml, corresponding to an approximately 10-fold median cytotoxic dose [CD50] of the batch used in this study) were added to each well of a 96-well culture plate and then incubated for 72 hr at 37°C. For the in vivo assays, each antibody diluted with PBS was reacted with 35 ng Stx2 (corresponding to a 5-fold median lethal dose [LD50] of the batch used in this study) in a 0.3 ml aliquot for 30 min at 37°C, and was then injected intraperitoneally into at least 5 female ICR mice (4 weeks of age; Japan SLC). The animals were observed for 1 week and their survival was recorded. All animal experiments were carried out in strict accordance with the recommendations of the Regulations for the Management of Laboratory Animals at Fujita Health University. All procedures were approved by the Institutional Animal Care and Use Committee of Fujita Health University (Permit Number: M2301).
Flow cytometry analysis. Seventy-two nanograms of Stx2 (corresponding to approximately 1 pmol), which was preincubated with a 10-fold amount (720 ng) of each mAb for 30 min, was added to 2×105 of HeLa229 cells. After reaction for 30 min, each cell sample was washed with PBS, and then reacted with 1 μg of anti-Stx2-His rabbit IgG which was purified from the antiserum (29) by rProtein A-SepharoseTM Fast Flow (GE Healthcare) for 30 min. After washing, the each cell sample was reacted with 0.5 μg of FITC-labeled anti-rabbit IgG goat F(ab’)2 (Santa Cruz, Dallas, TX) and then analyzed by FACSCaliburTM (Becton Dickinson, Franklin Lakes, NJ). All reaction steps were conducted on ice.
Western blot analysis. Protein samples were separated in a 15% polyacrylamide gel and electroblotted onto a
8
polyvinylidene difluoride (PVDF) membrane. After incubation in a blocking buffer for 1 hr, the membrane was reacted with each mAb or anti-Stx2-His rabbit serum (to detect the loading control reaction with each mStx2 expressed) diluted in PBS containing 5% bovine serum albumin for 1 hr, followed by a reaction with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG goat antibody (Jackson ImmunoResearch, West Grove, PA) or anti-rabbit immunoglobulin swine immunoglobulin (Dako, Copenhagen, Denmark), respectively. Specific bands were detected with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) using an LAS4000 image analyzer (Fujifilm, Tokyo, Japan). All reactions were carried out at room temperature, and the membrane was washed 3 times with T-PBS for 5 min before each reaction.
Determination of the sequences of the VH and VL regions of the mAbs. mRNA from each hybridoma was prepared with Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s method, and then each VH and VL region was amplified independently by RT-PCR using LA-Taq (Takara, Otsu, Japan) and specific primers (listed in Table S1). The amplified products were ligated into the pCR2.1 T-vector (Life Technologies, Carlsbad, CA) and then transformed into TOP10F′ competent cells (Life Technologies). Several colonies, which were picked up and cultured in Luria-Bertani broth containing ampicillin for several hours at 37°C, were used as the template for PCR with a primer set (M13-forward and M13-reverse), and then DNA sequencing was conducted using a Genetic Analyzer Avant3130xl (Applied Biosystems, Foster City, CA). The data were analyzed by Sequencher Ver.4.5 (Gene Codes, Ann Arbor, MI).
Expression of single chain FV-protein A fusion antibodies (scFV-PP) in E. coli. In order to confirm whether the determined sequences were correct, amplified products of each VH and VL gene fragment were ligated into the pSCCA5-E8d phagemid vector, which was modified from pAALSC (32), at the SfiI-NotI sites and NcoI-AscI sites,
9
respectively (as shown in Fig. S1; the nucleotide sequence of pSCCA5-E8d has been submitted to DDBJ under the accession no. AB969670). Furthermore, in order to fuse the double Fc binding domain gene of protein A to the 3′-end of the VL gene, the cp3 gene was eliminated after SalI digestion, and then the resultant plasmid was self-ligated. The E. coli DH12S strain was transformed with each scFV-PP expression plasmid and cultured in 2×YT broth containing ampicillin and 0.1% glucose at 30°C until the exponential growth phase. The expression of scFV-PP was induced by adding isopropyl β-D-1-thiogalactopyranoside at a final concentration of 1 mM and then cultured overnight at 30°C. After centrifugation, each scFV-PP was purified from the mixture of culture supernatant and periplasmic fraction which was extracted from bacterial cells (33) by using IgG-SepharoseTM 6 Fast Flow (GE Healthcare).
Evaluation of the reaction of scFV-PP with Stx2. In order to confirm the validity of the sequences of each mAb, ELISA and western blot analysis were conducted. In ELISA, 0.5 μg/50 μl Stx2 was plated in a 96-well plate and then incubated overnight at 4°C. After incubation with 100 μl blocking buffer for 90 min, the plate was reacted with 50 μl of each scFV-PP or anti-influenza virus-specific Fab-PP (F005-126, as a negative control) (34) diluted with blocking buffer for 1 hr. For reaction with 2×IgG binding domain of protein A (PP) fused at the C-terminal end of the Stx2-specific scFV, each well was reacted with 50 μl HRP-conjugated anti-rabbit immunoglobulin swine immunoglobulin (Dako) diluted with blocking buffer for 1 hr, followed by reaction with 50 μl of the substrate solution for HRP (citrate buffer [pH 5.0] containing 0.04% [wt/vol] o-phenylenediamine and 0.02% [vol/vol] H2O2) for 30 min. After stopping the reaction with 50 μl of 1 M H2SO4, absorbance at 490 nm was measured with a plate reader. In western blot, the cell extract containing Stx2 was blotted onto the PVDF membrane and then blocked in the same manner as described above. The membrane was reacted with each scFV-PP diluted with the blocking buffer, followed by reaction with HRP-conjugated anti-sheep IgG (H&L) rabbit antibody (Rockland Immunochemicals, Limerick, PA) through the interaction with PP of scFV.
10
Primer sequences. All primer sequences used in this study are listed in our previous papers (28, 29) and in Table S1 of this manuscript.
11
Results
Isolation of Stx2-specific mAb clones. In this study, we screened 4 clones that produced Stx2-His-specific antibodies using ELISA in the first trial (Fig. 1a). Since each mAb was screened with Stx2-His, we attempted to confirm the specificity of each mAb against non-tagged Stx2 in cell extracts by western blot analysis to exclude the possibility of a histidine-tag-specific reaction. mAbs 40 and 131 reacted only to Stx2A, whereas mAb 45 reacted only to Stx2B, indicating that clones 40 and 131 were Stx2A-specific mAb-producing clones, while clone 45 was an Stx2B-specific mAb-producing clone. Conversely, mAb 134 did not react with either the A or B bands in western blot analysis. In addition, since mAb 134 reacted to another histidine-tagged protein that we had prepared previously (data not shown), we concluded that mAb 134 was a histidine tag-specific antibody that was applicable as the negative control in this study. In the second trial, since we had previously developed an affinity purification method for the mStx2 antigen without the use of any tag proteins (29), we used an improved mStx2 (A:E167Q+R170L, B:D17N) as an antigen to avoid generating histidine tag antibody-producing clones, and obtained 4 Stx2A-specific (clones 59A1, 64E5, 68G11, and 70F5) and 4 Stx2B-specific (clones 61C6, 72F2, 73B4, and 75D9) mAb-producing clones (Fig. 1b). All antibodies obtained in this study were of the IgG1 heavy chain and kappa light chain classes.
In vitro neutralization effects of each mAb against Stx2. In order to analyze the neutralization activity of each mAb against Stx2, in vitro neutralization assays were conducted by using HeLa229 cells. For the 4 antibodies obtained in the first trial, Stx2B-specific mAb 45 showed the highest neutralization activity against Stx2 (Fig. 2a). Stx2A-specific mAb 131 also showed a higher neutralization effect than the other Stx2A-specific mAb (mAb 40) and the negative control (mAb 134),
12
although it was considerably weaker than mAb 45. Similarly, among the 8 antibodies obtained in the second trial, 4 different Stx2B-specific antibodies (61C6, 72F2, 73B4, and 75D9) showed a stronger neutralization effect against Stx2 than the 4 different Stx2A-specific antibodies (59A1, 64E5, 68G11, and 70F5; Fig. 2b); especially, mAb 75D9 showed the highest neutralization activity among them. Although some Stx2A-specific mAbs showed neutralization activity, their activity was lower than that of mAbs 45 and 75D9 in each trial. Therefore, we focused on the two mAb clones 45 and 75D9 and analyzed their in vivo neutralization activity.
Inhibitory effect of mAbs for Stx2 binding to HeLa229 cells. Since we hypothesized that higher in vitro neutralization activities of Stx2B-specific mAbs 45 and 75D9 than that of Stx2A-specific mAbs were related to inhibition of receptor binding, the binding inhibition of each mAb against Stx2 to HeLa229 cells was analyzed using flow cytometry analysis. As shown in Fig. 3, a ten-fold amount of Stx2B-specific neutralizing mAbs 45 and 75D9 completely inhibited the binding of 72 ng of Stx2, whereas that of negative control mAb 134 did not show any inhibition of Stx2 binding. Regarding the Stx2A-specific mAbs, although two kinds of neutralizing mAbs 131 and 70F5 also partially inhibited Stx2 binding and non-neutralizing mAb 64E5 did not, these results are not reproducible in some trials (data not shown). These results indicate at least that high neutralization activities of mAbs 45 and 75D9 against Stx2 are based on the complete inhibition of the receptor binding.
In vivo neutralization effects of mAbs against Stx2. Each Stx2A- and Stx2B-specific mAb which showed the highest in vitro neutralization activity in each trial was administered intraperitoneally to mice with Stx2. As shown in Table 1, 1000 ng of each Stx2B-specific mAbs (45 and 75D9) completely protected the mice from a 5-fold LD50 of Stx2, whereas that of each Stx2A-specific mAbs (131 and 70F5) partially protected. Especially, in the case of 75D9, a complete protection
13
effect was retained at 100 ng. mAb 134 (as a negative control) did not show any neutralization activity, even at 1000 ng. From the dose-dependent protection afforded by these antibodies, mAb 75D9 seemed to be the most effective antibody prepared in this study, leading to the correlation with the in vitro assays.
Identification of the amino acid residues of Stx2 associated with recognition by each mAb. In vitro and in vivo neutralization assays revealed that mAbs 45 and 75D9 are alternative candidates for the development of mouse-human chimeric Stx2-neutralizing antibodies. Therefore, in order to identify the amino acid residues in Stx2 that were recognized by mAbs 45 and 75D9, western blot analysis was conducted against the prepared mStx2 clones. The locations of the mutations introduced to the B subunit were selected to focus on the receptor binding sites (W29, S54, E57) (16, 35) and the different residues (K5, D16, D17, D24, K26, S31, K52, S54, E57, E64, Q66, N69, D70) from those in the other variant toxins (Stx2c, Stx2d, Stx2e, and Stx2f). As shown in Fig. 4a, both mAbs that neutralized the toxicity of Stx2 did not react to the single W29A, S54N, and E57S mutants, each amino acid residue of which is associated with receptor binding (16, 35). These results indicate that the Stx2B-specific mAbs 45 and 75D9 neutralize the toxicity of Stx2 by binding to the receptor binding sites of the B subunit as shown in Fig. 4b.
Amino acid sequences of the VH and VL regions of mAbs 45 and 75D9. Additionally, we compared the amino acid sequences of the VH and VL regions of mAbs 45 and 75D9 deduced from their nucleotide sequences. The sequences of the VH and VL regions have been submitted to the DDBJ under the accession nos. AB969666, AB969667, AB969668, and AB969669 (as Stx2-45-VH, Stx2-45-VL, Stx2-75-VH, and Stx2-75-VL, respectively). The validity of the sequences of each mAb was confirmed by the expression of the scFV-PP of each clone (scFV-PP 45, scFV-PP 75D9) in the E. coli DH12S strain. As shown in Fig. 5, we confirmed the reactivity of each scFV-PP against the Stx2B both in ELISA and
14
western blot analysis. The amino acid sequences of the variable region between both antibodies were quite different, indicating that each antibody was derived from a different clone.
15
Discussion In this study, we obtained 11 different Stx2-specific mAb-producing hybridomas following the intranasal immunization of mice with Stx2 antigens and mLT; 6 were Stx2A-specific and 5 were Stx2B-specific mAb-producing clones. Although mice tend to produce a small amount of Stx2B-specific antibody at the polyclonal antibody level (24), we reported previously that intranasal immunization of histidine-tagged Stx2B with mLT induced the production of Stx2B-specific antibodies that neutralized the toxicity of Stx2 in the sera of mice (25, 26). Although the reason why mLT induces different immunoreactivities against Stx2 by intranasal immunization is unknown, these results indicate that mucosal immunization with mLT might be a useful strategy to raise antibodies against antigens that cannot induce specific antibodies by parenteral (subcutaneous or intramuscular) routes. Although our ultimate purpose was to obtain the Stx2-specific mAbs which cross-reacted with Stx1, we confirmed that none of the mAbs cross-reacted with Stx1 in the early process of screening Stx2-specific mAb hybridoma clones (data not shown). In the neutralization assay using HeLa229 cells, the Stx2B-specific antibodies obtained in this study tended to show a higher neutralization effect than the Stx2A-specific mAbs against Stx2, and the highest neutralizing mAb clones (45 and 75D9) prepared in each trial showed strong protective effects on mice against a 5-fold LD50 Stx2 challenge. Nakao et al. reported that 50 % neutralizing concentration of anti-Stx2B mAb VTm1.1 against 125 pg/ml of Stx2 on ACHN cells was approximately 55 ng/ml (16). On the other hand, Edwards et al. reported that the amount of mAb 11E10 required to neutralize 1 CD50 of Stx2 (corresponding to 1 pg used in their study) on Vero cells was calculated at 2.7 ng (18). In the case of our mAb, 50 % neutralizing concentration of the mAb 75D9 against 100 pg/ml of Stx2 on HeLa229 cells was between 1.6-8 ng/ml (Fig. 2). These indicate that 75D9 has especially strong neutralization activity which is comparable to VTm 1.1 and 11E10, although the assay methods are different from each other.
16
The flow cytometry analysis using HeLa229 cells revealed that the mAbs 45 and 75D9 directly inhibited the binding of Stx2 to the receptor. Furthermore, neither Stx2B-specific mAbs reacted to 3 different B subunit mutants (W29A, S54N, and E57S). It was reported that VTm1.1 neutralized the toxicity of Stx2 by binding to amino acid E56 (corresponding to E57 in our study) in Stx2B (16). Since the crystal structure of Stx2 had not been reported at that time, they speculated upon the location of E57 in the B subunit by comparing it to the corresponding amino acid of Stx1, and suggested that E57 was associated with receptor binding (16). According to the crystal structure of Stx2 reported by Fraser et al. (35), E57 is located near W29, which is associated with receptor binding (Fig. 4b). Furthermore, they mentioned the association of S54 with receptor binding (35). These indicate that mAbs 45 and 75D9 neutralize the toxicity of Stx2 by binding to the receptor binding site of the B subunit. On the other hand, Stx2A-specific mAbs 131 and 70F5, which partially neutralized the toxicities of Stx2, partially inhibited the binding of Stx2 to HeLa229 cells. However, since these inhibitions were not reproducible in some trials, we could not discuss whether or not these Stx2A-specific mAbs partially neutralized the toxicity through physical inhibition of the receptor binding. As shown in Fig. 2, the neutralization activity of the remaining 3 Stx2B-specific mAbs (61C6, 72F2, and 73B4) prepared in the second trial was also at the same level as that of mAb 45 prepared in the first trial, and their reaction profiles against mStx2s in western blot analysis were also similar to mAbs 45 and 75D9 (data not shown). These results indicate that these antibodies neutralize toxicity by inhibiting receptor binding, and that the neutralization epitopes on the B subunit of Stx2 are limited to a specific region associated with receptor binding, as shown in Fig. 4b. In this study, we determined the nucleotide sequences of the VH and VL regions of mAbs 45 and 75D9 for the development of mouse-human chimeric Stx2-neutralizing antibodies. We confirmed the validity of the sequences of each mAb by Stx2-specific ELISA and western blot using each scFV-PP protein prepared. The reactivity of scFV-PP of each clone against Stx2 on western blot was lower than that of original mAb, due to the fact that the scFV is a monovalent
17
antibody. Additionally, since the principle of each method used in scFv-PP is somewhat different from that used in the original mAb from the point of using interaction of scFV-PP with secondary antibodies through the Fc region, we can only evaluate the validity of the each sequences in specificities, but not in sensitivities compared with the original mAb. We still have several different candidate mAbs that neutralize the toxicity of Stx2, including Stx2A-specific neutralization mAbs (61C6, 68G11, 72F2, 73B4). Even in the case that each mAb recognizes similar epitopes, their neutralization effect against the toxin is also dependent on their affinity to the neutralization epitopes. Although mAbs 45 or 75D9 show a sufficient neutralization effect even in independently conducted trials, we need to investigate the combination effects of our mAbs on effective Stx2-neutralization in the near future.
18
Acknowledgements
This work was supported, in part, by JSPS KAKENHI (Number 24590539), the Strategic Research Base Development Program for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and Health and Labor Sciences Research Grants for Research on Global Health Issues from the Ministry of Health, Labor, and Welfare, Japan.
Disclosure The authors declare no conflicts of interest or financial support.
19
References
1
Tarr P.I., Gordon C.A.,Chandler W.L. (2005) Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet
2
365: 1073-86.
Karmali M.A. (2004) Infection by Shiga toxin-producing Escherichia coli: an overview. Mol Biotechnol 26: 117-22.
3
O'Brien A.D., Tesh V.L., Donohue-Rolfe A., Jackson M.P., Olsnes S., Sandvig K., Lindberg A.A.,Keusch G.T. (1992) Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis. Curr Top Microbiol Immunol
4
180: 65-94.
Boerlin P., McEwen S.A., Boerlin-Petzold F., Wilson J.B., Johnson R.P.,Gyles C.L. (1999) Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. J Clin Microbiol
37:
497-503. 5
Ostroff S.M., Tarr P.I., Neill M.A., Lewis J.H., Hargrett-Bean N.,Kobayashi J.M. (1989) Toxin genotypes and plasmid profiles as determinants of systemic sequelae in Escherichia coli O157:H7 infections. J Infect Dis
160:
994-8. 6
Kleanthous H., Smith H.R., Scotland S.M., Gross R.J., Rowe B., Taylor C.M.,Milford D.V. (1990) Haemolytic uraemic syndromes in the British Isles, 1985-8: association with verocytotoxin producing Escherichia coli. Part 2: Microbiological aspects. Arch Dis Child
7
65: 722-7.
Scotland S.M., Willshaw G.A., Smith H.R.,Rowe B. (1987) Properties of strains of Escherichia coli belonging to serogroup O157 with special reference to production of Vero cytotoxins VT1 and VT2. Epidemiol Infect
99:
613-24. 8
Takeda Y., Kurazono H.,Yamasaki S. (1993) Vero toxins (Shiga-like toxins) produced by enterohemorrhagic Escherichia coli (verocytotoxin-producing E. coli). Microbiol Immunol
37: 591-9.
20
9
Tesh V.L., Burris J.A., Owens J.W., Gordon V.M., Wadolkowski E.A., O'Brien A.D.,Samuel J.E. (1993) Comparison of the relative toxicities of Shiga-like toxins type I and type II for mice. Infect Immun
61:
3392-402. 10
Zhang X., McDaniel A.D., Wolf L.E., Keusch G.T., Waldor M.K.,Acheson D.W. (2000) Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. J Infect Dis
11
Matsushiro A., Sato K., Miyamoto H., Yamamura T.,Honda T. (1999) Induction of prophages of enterohemorrhagic Escherichia coli O157:H7 with norfloxacin. J Bacteriol
12
181: 2257-60.
Walterspiel J.N., Ashkenazi S., Morrow A.L.,Cleary T.G. (1992) Effect of subinhibitory concentrations of antibiotics on extracellular Shiga-like toxin I. Infection
13
20: 25-9.
Wong C.S., Jelacic S., Habeeb R.L., Watkins S.L.,Tarr P.I. (2000) The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N Engl J Med
14
181: 664-70.
342: 1930-6.
Mukherjee J., Chios K., Fishwild D., Hudson D., O'Donnell S., Rich S.M., Donohue-Rolfe A.,Tzipori S. (2002) Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection. Infect Immun
15
70: 612-9.
Sheoran A.S., Chapman S., Singh P., Donohue-Rolfe A.,Tzipori S. (2003) Stx2-specific human monoclonal antibodies protect mice against lethal infection with Escherichia coli expressing Stx2 variants. Infect Immun 71: 3125-30.
16
Nakao H., Kiyokawa N., Fujimoto J., Yamasaki S.,Takeda T. (1999) Monoclonal antibody to Shiga toxin 2 which blocks receptor binding and neutralizes cytotoxicity. Infect Immun
17
67: 5717-22.
Perera L.P., Marques L.R.,O'Brien A.D. (1988) Isolation and characterization of monoclonal antibodies to Shiga-like toxin II of enterohemorrhagic Escherichia coli and use of the monoclonal antibodies in a colony enzyme-linked immunosorbent assay. J Clin Microbiol
18
26: 2127-31.
Edwards AC M.-C.A., Arbuthnott K, Stinson JR, Schmitt CK, Wong HC, O'Brien A.D. (1998) Vero cell neutralization and mouse protective efficacy of humanized monoclonal antibodies against Escherichia coli toxins
21
Stx1 and Stx2, p 388–392. In Kaper JB, O'Brien AD, editors (ed), Escherichia coli O157:H7 and other Shiga toxin-producing E coli strains ASM Press, Washington, DC. 19
Kimura T., Co M.S., Vasquez M., Wei S., Xu H., Tani S., Sakai Y., Kawamura T., Matsumoto Y., Nakao H.,Takeda T. (2002) Development of humanized monoclonal antibody TMA-15 which neutralizes Shiga toxin 2. Hybrid Hybridomics
20
21: 161-8.
Yamagami S., Motoki M., Kimura T., Izumi H., Takeda T., Katsuura Y.,Matsumoto Y. (2001) Efficacy of postinfection treatment with anti-Shiga toxin (Stx) 2 humanized monoclonal antibody TMA-15 in mice lethally challenged with Stx-producing Escherichia coli. J Infect Dis
21
184: 738-42.
Bitzan M., Poole R., Mehran M., Sicard E., Brockus C., Thuning-Roberson C.,Rivière M. (2009) Safety and pharmacokinetics of chimeric anti-Shiga toxin 1 and anti-Shiga toxin 2 monoclonal antibodies in healthy volunteers. Antimicrob Agents Chemother
22
53: 3081-7.
Dowling T.C., Chavaillaz P.A., Young D.G., Melton-Celsa A., O'Brien A., Thuning-Roberson C., Edelman R.,Tacket C.O. (2005) Phase 1 safety and pharmacokinetic study of chimeric murine-human monoclonal antibody cαStx2 administered intravenously to healthy adult volunteers. Antimicrob Agents Chemother
23
49: 1808-12.
López E.L., Contrini M.M., Glatstein E., González Ayala S., Santoro R., Allende D., Ezcurra G., Teplitz E., Koyama T., Matsumoto Y., Sato H., Sakai K., Hoshide S., Komoriya K., Morita T., Harning R.,Brookman S. (2010) Safety and pharmacokinetics of urtoxazumab, a humanized monoclonal antibody, against Shiga-like toxin 2 in healthy adults and in pediatric patients infected with Shiga-like toxin-producing Escherichia coli. Antimicrob Agents Chemother
24
54: 239-43.
Wen S., Teel L., Judge N.,O'Brien A.D. (2006) Genetic toxoids of Shiga toxin types 1 and 2 protect mice against homologous but not heterologous toxin challenge. Vaccine
25
24: 1142-48.
Tsuji T., Shimizu T., Sasaki K., Shimizu Y., Tsukamoto K., Arimitsu H., Ochi S., Sugiyama S., Taniguchi K., Neri P.,Mori H. (2008) Protection of mice from Shiga toxin-2 toxemia by mucosal vaccine of Shiga toxin 2B-His with Escherichia coli enterotoxin. Vaccine
26
26: 469-76.
Tsuji T., Shimizu T., Sasaki K., Tsukamoto K., Arimitsu H., Ochi S., Taniguchi K., Noda M., Neri P.,Mori H.
22
(2008) A nasal vaccine comprising B-subunit derivative of Shiga toxin 2 for cross-protection against Shiga toxin types 1 and 2. Vaccine 27
26: 2092-9.
Connell T.D. (2007) Cholera toxin, LT-I, LT-IIa and LT-IIb: the critical role of ganglioside binding in immunomodulation by type I and type II heat-labile enterotoxins. Expert Rev Vaccines
28
Arimitsu H., Sasaki K., Shimizu T., Tsukamoto K., Shimizu T.,Tsuji T. (2013) Large-scale preparation of Shiga toxin 2 in Escherichia coli for toxoid vaccine antigen production. Microbiol Immunol
29
57: 38-45.
Arimitsu H., Sasaki K., Kojima H., Yanaka T.,Tsuji T. (2013) Simple Method for Shiga Toxin 2e Purification by Affinity Chromatography via Binding to the Divinyl Sulfone Group. PLoS One
30
6: 821-34.
8: e83577.
Tsuji T., Yokochi T., Kamiya H., Kawamoto Y., Miyama A.,Asano Y. (1997) Relationship between a low toxicity of the mutant A subunit of enterotoxigenic Escherichia coli enterotoxin and its strong adjuvant action. Immunology
31
90: 176-82.
Neri P., Nagano S.I., Yokoyama S., Dohi H., Kobayashi K., Miura T., Inazu T., Sugiyama T., Nishida Y.,Mori H. (2007) Neutralizing activity of polyvalent Gb3, Gb2 and galacto-trehalose models against Shiga toxins. Microbiol Immunol
32
51: 581-92.
Iba Y., Ito W.,Kurosawa Y. (1997) Expression vectors for the introduction of highly diverged sequences into the six complementarity-determining regions of an antibody. Gene
33
Skerra A.,Plückthun A. (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science
34
194: 35-46.
240: 1038-41.
Iba Y., Fujii Y., Ohshima N., Sumida T., Kubota-Koketsu R., Ikeda M., Wakiyama M., Shirouzu M., Okada J., Okuno Y., Kurosawa Y.,Yokoyama S. (2014) Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses. J Virol
35
88: 7130-44.
Fraser M.E., Fujinaga M., Cherney M.M., Melton-Celsa A.R., Twiddy E.M., O'Brien A.D.,James M.N. (2004) Structure of Shiga Toxin Type 2 (Stx2) from Escherichia coli O157:H7. J Biol Chem
279: 27511-7.
23
Figure Legends
Fig. 1. Immunological properties of the mAbs.
Antibody level of mAbs obtained by immunizing Stx2-His toxoid in
the first trial (a) and by immunizing mStx2 in the second trial (b). Stx2-His was used as an immobilized antigen of ELISA in each trial. The data represent the mean of three separate experiments and the error bars indicate SEM. The data of the B subunit-specific antibodies are represented as closed color symbols. The western blot analysis of each mAb to 5 μl of cell extract from non-tagged Stx2-expressing E. coli MV1184 is shown on the right panel of each trial. Each lane was separated after blocking, and combined again in the detection step. The concentration of each mAb in western blot analysis was as follows:
40 (1 μg/ml); 45 (3 μg/ml); 131 (2.5 μg/ml); 134 (1 μg/ml); 59A1 (10 ng/ml); 61C6 (1 μg/ml);
64E5 (5 ng/ml); 68G11 (500 ng/ml); 70F5 (2.5 μg/ml); 72F2 (1 μg/ml); 73B4 (1 μg/ml); 75D9 (1 μg/ml).
Fig. 2. In vitro neutralization activity of each mAb against Stx2. In the first (a) and second (b) trials. The data represent the mean of three separate experiments and the error bars indicate SEM. The data of the B subunit-specific antibodies are represented as closed color symbols.
Fig. 3. Inhibitory effect of mAbs against Stx2 binding to HeLa229 cells. Stx2 (72 ng) was preincubated with 10-fold amount (720ng) of each mAb, and then added to 2×105 of the cells. Binding level of toxins on the cell surface was detected by anti-Stx2-His rabbit IgG, followed by FITC-labeled anti-rabbit IgG goat F(ab’) 2. Open black histgrams; untreated cells, shaded histgrams; Stx2-treated cells, open colored histgrams; Stx2-mAb mixture-treated cells.
24
Fig. 4. Identification of the amino acid residues of Stx2B associated with the binding of mAbs 45 and 75D9. (a) Western blot analysis of the mAbs 45 (3 μg/ml) and 75D9 (1 μg/ml) against 5 μl cell extract containing each mStx2 with a mutation in the B subunit. Loading control reactions against each mStx2 were detected by anti-Stx2-His rabbit serum (polyclonal). The mutated positions are shown at the bottom of the panel. (b) Location of the amino acid residues on the B subunit pentamer associated with the binding of mAbs 45 and 75D9. Diagonal bottom view of the crystal structure of Stx2 modified from PDB ID 1R4P, reported by Fraser et al. (35). The residues assessed are shown in blue (W29), green (S54), and red (E57). The A subunit is shown in gray.
Fig. 5. Reactivities of the scFV-PP 45 and scFV-PP 75D9 against Stx2. a) Each diluted scFV-PP, which was purified from DH12S transformed with the scFV-PP expression plasmid of each Stx2B-specific mAb clone, was reacted with 0.5 μg/50 μl of Stx2, followed by reaction with HRP-conjugated anti-rabbit immunoglobulin swine immunoglobulin (Dako). Purified influenza virus-specific Fab-PP was used as a negative control in this assay. b) The western blot analysis of each scFV-PP to the cell extract from non-tagged Stx2-expressing E. coli MV1184.
Fig. S1. Schematic diagram of the single chain gene cloning site of the scFV-PP expression plasmid pSCCA5-E8d.
Table S1. Oligonucleotide primers used in this study.
25
List of Abbreviations
CD50, median cytotoxic dose EHEC, Enterohemorrhagic Escherichia coli ELISA, enzyme-linked immunosorbent assay HRP, horseradish peroxidase HUS, hemolytic uremic syndrome LD50, median lethal dose mAb, monoclonal antibody mLT, mutant heat-labile enterotoxin mStx2, mutant Shiga toxin 2 PBS, phosphate-buffered saline Stx2, Shiga toxin 2 Stx2A, Shiga toxin 2 A subunit Stx2B, Shiga toxin 2 B subunit
26
Table 1. In vivo neutralization activity of Stx2-specific mAbs against Stx2†‡ Amonut of mAb (ng) mixed with 35ng Stx2 Clone No. 1,000
100
50
30
10
45
5/5
3/5
2/5
ND
0/5
75D9
5/5
5/5
ND
3/5
1/5
131
2/5
1/5
ND
ND
ND
70F5
2/5
0/5
ND
ND
ND
134
0/5
ND
ND
ND
ND
†
35 ng (corresponds to 5-fold LD50) of Stx2 were inoculated intraperitoneally with each diluted mAb.
‡
Result is shown as the number of survived / challenged mice.
ND: Not determined
27
Figure 1
28
Figure 2
29
Figure 3
30
Figure 4
31
Figure 5
