Monoclonal antibody against the poly-γ-d -glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Biochimica et Biophysica Acta 1830 (2013) 2804–2812

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Monoclonal antibody against the poly-γ-D-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge Jeyoun Jang a, 1, Minhui Cho a, 1, Hae-Ri Lee a, Kiweon Cha a, Jeong-Hoon Chun a, Kee-Jong Hong a, Jungchan Park b, c, Gi-eun Rhie a,⁎ a b c

Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 187 Osongsaengmyeong2-ro, Cheongwon-gun, Chungbuk 363-951, Republic of Korea Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea Protein Research Center for Bioindustry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea

a r t i c l e

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Article history: Received 2 April 2012 Received in revised form 2 November 2012 Accepted 10 November 2012 Available online 28 November 2012 Keywords: Bacillus anthracis Monoclonal antibody Modeling Lethal toxin Poly-γ-D-glutamic acid capsule

a b s t r a c t Background: The poly-γ-D-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT. Methods: To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with Kd values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed. Results: Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen. General significance: Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Anthrax is a highly lethal infectious disease caused by the sporeforming bacterium Bacillus anthracis [1]. Exotoxin, the major virulence factor of B. anthracis, is composed of three distinct proteins—protective antigen (PA), edema factor (EF), and lethal factor (LF)—that are secreted individually as non-toxic monomers [1]. The binding of LF or EF to PA results in the formation of active lethal toxin (LT) or edema toxin, respectively [1]. LF is a metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinase (MAPKK; [2]), which plays an intermediate role in the activation of MAP kinase (MAPK) signaling Abbreviations: EF, Edema factor; HRP, Horseradish peroxidase; i.p., Intraperitoneal; LF, Lethal factor; LT, Lethal toxin; MAPK, MAP kinase; MAPKK, Mitogen-activated protein kinase kinase; mAb, Monoclonal antibody; PGA, Poly-γ-D-glutamic acid; PA, Protective antigen; 3D, Three dimensional ⁎ Corresponding author. Tel.: +82 43 719 8270; fax: +82 43 719 8309. E-mail address: [email protected] (G. Rhie). 1 These authors contributed equally to this work. 0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2012.11.006

pathways [3]. LF promotes macrophage death by disrupting the MAPKdependent pathways that regulate pro-survival genes [3,4] and by activating proteasome- and inflammasome-dependent pathways that cleave the inflammasome component, NALP1b and activate caspase-1 [5,6]. EF is a calmodulin-dependent adenylate cyclase that causes a prolonged increase in cytosolic cyclic adenosine monophosphate, which triggers an efflux of fluid from the host cell, resulting in localized edema [7]. B. anthracis contains another virulence factor, the capsule, which is composed of poly-γ-D-glutamic acid (PGA) [8]. The weakly immunogenic and anti-phagocytic PGA capsule protects B. anthracis from immune surveillance through a mechanism that is similar to that of the capsular polysaccharides that protect other pathogens, such as pneumococci and meningococci, from phagocytosis [8]. A recent study showed that degradation of PGA enhances in vitro macrophage phagocytosis and neutrophil killing of encapsulated B. anthracis, further supporting the anti-phagocytic nature of the capsule [9]. In a mouse model of pulmonary anthrax, the PGA capsule of B. anthracis was

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shown to be essential for dissemination from the lungs and for persistence and survival of the bacterium in the host [10]. Virulence appears to be associated with anti-phagocytic properties of the capsule [11–13]. In addition, the capsule released from B. anthracis has been reported to be associated with LT in the blood of experimentally infected animals [14], and PGA enhances the cytotoxic effect of LT on J774A.1 cells in a concentration-dependent manner [15]. PGA also augments the death of mice challenged with LT, which indicates that PGA in combination with LT acts to intensify the toxemia that occurs at the terminal stage of anthrax infection [15]. These reports emphasize the importance of the PGA capsule in the pathogenesis of anthrax infection. In addition to PA, the PGA capsule has been used as a potential target for development of vaccines and neutralizing mAbs [16–19]. Both active and passive vaccination targeting the B. anthracis capsule protects animals against experimental infection, suggesting that methods to increase phagocytosis of encapsulated B. anthracis bacilli may be valuable in preventing, inhibiting progression, and treating anthrax. In this study, we produced mAbs against PGA and their three dimensional (3D) homology models with cognitive PGA complex were analyzed. Produced anti-PGA mAbs showed their protective role in preventing the progress of anthrax disease by inhibiting the enhanced cytotoxic activity of LT by PGA as well as by eliminating the vegetative form of encapsulated B. anthracis through specific interaction with PGA capsule. 2. Methods

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Hybridoma cell lines were cloned by limiting dilution. mAb-secreting hybridoma cell lines were grown and then injected into i.p. cavities of BALB/c mice that had been pretreated with 0.5 ml incomplete Freund's adjuvant 4 h prior to injection of hybridoma cells. Hybridoma cells were injected (1 × 10 6 cells/mouse) in 0.5 ml of PBS (pH 7.4). Mice usually developed ascitic fluid 10–14 days after inoculation. Ascites fluid was harvested and pooled from several mice for subsequent isolation of the mAbs via affinity chromatography on protein G (Pierce). 2.4. Sequencing and analysis of variable regions Total RNA was prepared from murine hybridoma cells with an RNeasy Mini kit (Qiagen). First-strand cDNA was synthesized from total RNA using Superscript III reverse transcriptase (Invitrogen) and an Ig 3′ constant region degenerative primer (Mouse Ig-Primer set; Novagen) according to the manufacturer's protocol. The cDNA was amplified by PCR using a series of Ig 5′ degenerative leader primers (Novagen). The PCR products were ligated into the pCR 2.1-TOPO vector (Invitrogen), cloned, and sequenced at Macrogen (Korea). The nucleotide sequences of heavy- and light-chain variable regions were analyzed using the IMGT/LIGM-DB comprehensive database of Ig and T-cell receptor nucleotide sequences with IMGT/ V-QUEST and IMGT/Junction Analysis tools. Variable regions were assigned to gene families based on molecular subgroup designations complied by IMGT (http://imgt.cines.fr).

2.1. Bacterial strains, culture, and isolation of PGA 2.5. Binding analysis by surface plasmon resonance (SPR) measurements For PGA production, Bacillus licheniformis ATCC 9945a, which produces a capsule composed of γ-linked glutamic acid residues [16], was used. PGA was purified from the supernatant of B. licheniformis cultures as described [15,20]. Because the average molecular mass of native PGA from B. licheniformis ATCC 9945a was approximately 500 kDa, PGA that had been fragmented to a molecular mass of b30 kDa (as described by [15] and [20]) was used for experiments. Fragmented PGA was verified by NMR spectroscopy (data not shown). B. anthracis H9401 is a clinical isolate from a Korean cutaneous anthrax patient and was grown in brain heart infusion (BHI) medium (Difco Laboratories). B. anthracis H9401 is fully virulent and possesses both pXO1 and pXO2 plasmids [20]. For mAb production and immunofluorescence analysis, pXO1 or pXO2 of B. anthracis H9401 was cured as described by Green et al. [21]. 2.2. Reagents PA purified as described [22] was obtained from Green Cross Co. (Korea). LF was obtained from List Biological Laboratories. Antibodies against α-tubulin and MAPKK4 were purchased from Cell Signaling, anti-caspase-1 was from Santa Cruz Biotechnology, anti-MAPKK2 was from BD Transduction Laboratories, and anti-MAPKK6 was from Upstate Biotechnology. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were purchased from Invitrogen and GE Healthcare, respectively. 2.3. mAb production BALB/c mice were immunized by intraperitoneal (i.p.) injection of heat-killed B. anthracis H9401 (pXO1−, pXO2 +) emulsified in complete Freund's adjuvant (1:1, Sigma-Aldrich). Mice were given booster immunizations (i.p.) with heat-inactivated B. anthracis H9401 (pXO1 −, pXO2 +) emulsified in incomplete Freund's adjuvant (1:1, Sigma-Aldrich) on days 14, 28, and 32 after the primary immunization, and spleens were collected 4 days later for production of hybridomas. Hybridomas were produced by fusion with the X63-Ag8.653 cell line using standard techniques [23]. Production of anti-PGA mAb by the hybridomas was evaluated by ELISA [20].

To determine binding affinity of antibodies, SPR measurements were conducted using a BIAcore 3000 (BIAcore). Purified mAbs A-11 and A-12 were directly immobilized on the dextran surfaces of a CM-5 chip (GE Healthcare Life-sciences) with standard coupling chemistry using 1-ethyl-3-(3-dimethylaminopropyl)-1-carbodiimide hydrochloride (0.2 M) and N-hydroxysuccinimide (0.05 M) in 10 mM sodium acetate (pH 5.5) to reach a target of approximately 500–1000 SPR response units (1 response unit = 1 pg protein/mm2). Then, 1 M ethanolamine-HCl, pH 8.5, was flowed over the surfaces to deactivate any residually reactive sites. For the binding assay, different concentrations of chemically synthesized 10-mer PGA peptides (Peptron) were flowed over mAb A-11 or A-12 at a rate of 5 μl/min in 50 mM sodium phosphate buffer (pH 5.8) with 0.005% (v/v) Tween-20 at 25 °C. A solution of 2 M NaCl was used as a regeneration buffer between runs. SPR data were analyzed using BIA evaluation software version 3.0 (BIAcore). 2.6. Immunofluorescence microscopy B. anthracis strains were cultured on capsule-producing agar plates (BHI agar supplemented with 0.7% sodium bicarbonate) at a concentration sufficient to yield confluent growth after one night at 37 °C in 5% CO2. For immunofluorescence, bacilli were incubated at 37 °C in a dilution of mAb for 1.5 h. After washing with PBS, the bacilli were incubated at 37 °C for 1.5 h with goat anti-mouse IgG conjugated to FITC (Santa Cruz Biotechnology). After washing, slides were examined with an Olympus Fluoview FV 1000 microscope. IgG (Sigma-Aldrich) was used as a negative control. 2.7. Opsonophagocytosis assay HL-60 cells (promyelocytic leukemia cells; CCL240; American Type Culture Collection, Rockville, MD USA) were used as the effector cells. Cells were grown and differentiated into granulocytes according to Romero-Steiner et al. [24]. Spores of B. anthracis H9401 were incubated in BHI broth supplemented with 0.7% sodium bicarbonate for 3 h in an incubator containing 20% CO2 for germination. For the functional assay, 20 μl of each mAb that was serially diluted in opsonophagocytosis buffer

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(Hank's balanced salt solution with Ca2+ and Mg2+, GIBCO) was placed in each well of a microtiter plate (Costar, Cambridge, MA USA) and 20 μl of appropriately diluted bacterial suspension (~1000 c.f.u.) in opsonophagocytosis buffer was added to each well. The assay plate was incubated at 37 °C in a 5% CO2 atmosphere for 15 min. Then, 20 μl of human complement source (Sigma) was added to each well, and 40 μl of washed, differentiated HL-60 cells (4×105) was immediately added to each well. The assay plate was incubated at 37 °C for 45 min with horizontal shaking (220 rpm) in room air to promote the phagocytic process. A 10 μl aliquot from each well was plated onto BHI agar plates, and the plates were incubated at 37 °C in 5% CO2 overnight. Viable colonies were counted, and the percentage of bacterial killing was calculated for each antibody titration by comparing with the number of colonies on control reference plates. All tests were performed in duplicate. 2.8. Effect of PGA on LT-mediated cytotoxicity LT-mediated cytotoxicity experiments were performed with mouse bone marrow-derived macrophages (BMDMs), which were purified and cultured according to the basic method of Stanley [25]. BMDM monolayers in Dulbecco's modified Eagle medium containing 10% FBS were cultured at 37 °C in 96-well plates (SPL Plastic Labware) up to a concentration of 1.5 × 10 5 cells/well. Prior to adding reagents to the cells, PGA was diluted with medium lacking 10% FBS and added to a set of 96-well plates at final concentrations of 0 (medium alone), 31.3, 62.5, 125, 250, and 500 μg/ml. PA was added to the PGA dilutions at final concentrations of 0.5 and 1.0 μg/ml, and LF was added at final concentrations of 0.1 and 0.2 μg/ml. The mixtures were incubated for 1 h at 37 °C before adding to BMDMs. Medium was removed from the BMDM monolayers, and 100 μl of the mixtures containing PGA, PA, and LF was added to the cells. For inhibition experiments, PGA (125 μg/ml) and LT (1.0 μg/ml of PA and 0.2 μg/ml of LF) were pre-incubated with various concentrations of mAb A-12 (3.75, 1.875, 0.937, or 0.468 μg/ml) for 1 h at 37 °C before adding to BMDMs. After 4-h incubation at 37 °C in 5% CO2, 100 μl of 3-[4,5-dimethylthylthiazol-2-yl]-2,5-dimethyl-tetrazolium bromide (MTT; Sigma-Aldrich) was added to each well at a final concentration of 0.5 mg/ml. After an additional 1-h incubation at 37 °C, the BMDMs were lysed by adding 100 μl extraction buffer (90% isopropyl alcohol containing 25 mM HCl and 0.5% [w/v] SDS). Absorbance was then measured at 570 nm with an ELISA reader (Tecan). For each assay, controls consisted of four wells that received only LT and four wells that contained only culture medium. Each sample was tested in duplicate and averaged for analysis. 2.9. Western blot analysis Cell pellets were resuspended in 100 μl 1 × bacterial protein extraction solution (Intron) supplemented with 1 × protease inhibitor cocktail (Sigma-Aldrich) and incubated on ice for 30 min to produce cell lysates. The cell lysates were clarified by microcentrifugation, and 30 μg of each clarified lysate was electrophoresed on a 12% NuPage Bis-Tris gel (Invitrogen). The separated proteins were transferred to 0.45-μm nitrocellulose membranes (Bio-Rad). Blots were blocked for 1 h in 100 mM Tris–HCl (pH 7.5), 0.9% NaCl, and 0.05% Tween-20 (TBS-T) containing 5% skim milk. Blots were then rinsed three times in TBS-T for 5 min and subsequently incubated with primary antibodies at dilutions specified by each manufacturer: rabbit anti-caspase-1, 1:1000; rabbit anti-α-tubulin, 1:1000; mouse anti-MAPKK2, 1:2500; rabbit anti-MAPKK4, 1:1000; and rabbit anti-MAPKK6, 1:1000. HRP-conjugated goat anti-rabbit IgG (1:5000) and HRP-conjugated goat anti-mouse IgG (1:5000) were used as secondary antibodies. After three washes with TBS-T, the blots were developed with enhanced chemiluminescence substrate (Pierce) and exposed to X-ray film (XAR5, Eastman Kodak).

2.10. Mouse challenge To verify the effect of PGA in vivo, 6-week-old female BALB/c mice (Orient Bio Inc.) were given tail vein injections of PGA (500 μg) only or PGA with LT (48 μg PA and 20 μg LF). For the protection experiment with anti-PGA mAb against PGA with LT, A-12 in 0.2 ml PBS (0.5 or 1.0 mg) was injected into the i.p. cavities of mice 4 h before the tail vein injection of LT and 500 μg of PGA. For the protection experiment with anti-PGA mAb against spore challenge, A-12 in 0.2 ml of PBS (0.5 or 1.0 mg) was injected into i.p. cavities of 6-week-old female A/J mice (Central Lab. Animal Inc.) 4 h before i.p. injection of 10× LD50 of B. anthracis H9401 [15]. Mice were observed for 14 days. Animals that survived for 14 days after the challenge were considered survivors. Animal study protocol (KCDC-12-036-1A) was approved by the Institutional Animal Care and Use Committee of the Korea National Institute of Health to avoid pain and distress. The housing and care of mice were in compliance with all relevant guidelines and requirements of the Institutional Animal Care and Use Committee of the Korea National Institute of Health. Animals were housed in specific pathogen free facilities and for challenge experiments, mice were moved to BL3 facility of Korea National Institute of Health. 2.11. Homology modeling of mAbs and docking complex analysis Homology modeling for A-11 and A-12 mAbs was performed by homology modeling programs SWISSMODEL [26] and MODELLER 9v9 (http://www.salilab.org/modeller/). By comparative modeling approach, PDB ID: 2UZI (murine mAb against ras protein) was selected as the most suitable template for modeling from Brookheaven PDB. The CDRs in target mAbs were identified from alignment with PDB ID: 2UZI. Final homology-based 3D models were used to construct docking complex models by Dock6 software (http://dock.compbio.ucsf.edu). Docking was carried out allowing full rotation and flexibility for the antigen while keeping mAb positions fixed in space. We used predefined 3D grids of the target protein with built-in search algorithm. 3D tri-γ-D-glutamic acid structure, as a representative PGA, was generated by AMBER program (http://amber.org) prior to docking simulation. To prepare for docking procedure, following steps were performed: (i) generation of pdb coordinates of tri-γ-D-glutamic acid and mAbs; (ii) calculation of charges; (iii) addition of hydrogen atoms; (iv) location of CDR pockets; (v) refinement of the complex. Interactive visualization and analysis of molecular structures were carried out by Pymol viewer or Chimera program (http://www.ucsf.edu/chimera). 2.12. Statistical analysis Differences in mean survival time after LT and/or PGA or spore injections were determined with the Kaplan–Meier Log-Rank test using SAS software (SAS Institute Inc., Cary, NC, USA). For other measures, the means ± standard deviation (SD) of treatment groups were compared to appropriate controls to determine statistical significance using a two-tailed Student's t-test. Differences were considered significant if P b 0.05. 3. Results 3.1. Production and characterization of PGA-specific mAbs Two PGA-specific mAbs, A-11 and A-12, were produced from mice immunized with heat-killed encapsulated B. anthracis H9401. The mAbs and the molecular features of the mAbs based on analysis of variable region sequences are identified in Table 1. Both mAbs belong to the IgG2 subclass (Table 1), and the competitive-inhibition ELISA showed that the two mAbs bound specifically to PGA (data not shown). SPR analysis showed that these mAbs specifically bound a chemically synthesized 10-mer of PGA with Kd values of 0.8 μM

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Table 1 Characteristics of mAbs. mAb

A-11 A-12

IgG subclass

IgG2 IgG2

VH IMGT subgroup

IGHV2-4-1*01 IGHV10-3*03

VH family

Vh2 Vh10

JH family

4 4

D REGION reading frame

2 0

(A-11) and 2.6 μM (A-12) (Table 1). A study by Chen et al. [27] showed that the Kd value derived from the 10-mer of PGA is an effective Kd that represents the binding strength of IgG to PGA in nature because the 10-mer peptide contains more than one antibodybinding site. Our Kd values (0.8 and 2.6 μM for A-11 and A-12, respectively) were 3–4 order of magnitude higher than those reported for anti-PGA chimpanzee mAbs (ranging 0.1–0.3 nM determined by fluorescence anisotropy; [27]) but were similar to those reported for anti-PGA mouse mAbs (0.36–19 μM determined by fluorescence perturbation [19]). 3.2. Specific binding of anti-PGA mAbs to encapsulated B. anthracis and their opsonophagocytic activity The ability of PGA-specific mAbs to bind encapsulated bacilli was examined. B. anthracis H9401 was grown on capsule-producing agar plates, and capsule production was confirmed by India ink staining [20]. As shown in Fig. 1, mAbs A-11 and A-12 completely coated the bacterial cells (Fig. 1A, B, E, & F). These antibodies did not bind a pXO2 cured strain of B. anthracis H9401 (Fig. 1C, D, G, & H), which does not produce a capsule. The IgG control did not react with the PGA capsule (data not shown). These results implicate that the PGA-specific mAbs, A-11 and A-12, can opsonize encapsulated B. anthracis cells. After binding to the capsule, antibodies can induce bacterial killing via complement-driven lysis and/or opsonophagocytosis [28]. To test the potency of anti-PGA mAbs, an opsonophagocytosis assay using A-11 and A-12 was performed with B. anthracis H9401 spores, a human complement source, and HL-60 cells that had been differentiated into granulocytes. As shown in Fig. 2, opsonophagocytosis potency correlated with mAb concentration. The addition of 160 μg of A-11 and A-12 significantly decreased the viability of bacilli by 60% and 90%, respectively, compared to control without antibody fraction. The IgG control did not show significant opsonophagocytosis activity (Fig. 2). These findings demonstrated that mAbs to PGA can kill bacilli by specific antibody-mediated opsonophagocytosis, suggesting that antibodies against the PGA capsule can be developed as therapeutics for treatment of anthrax by preventing the progress of the disease. 3.3. PGA treatment enhances the cytotoxic activity of LT on mouse BMDMs Previously, we reported that PGA enhances the cytotoxic effect of LT on J774A.1 cells [15]. To confirm the effect of PGA on the cytotoxic activity of LT on mouse primary cells, BMDMs were isolated and treated with various concentrations of PGA and/or LT, and cell viability was assessed after 4-h incubation using the MTT assay. The LT consisted of either 0.5 μg/ml PA and 0.1 μg/ml LF or 1.0 μg/ml PA and 0.2 μg/ml LF. As reported previously [15], treatment of BMDMs with LT caused cell death, and rates of cell death increased with increasing PA and LF concentrations (Fig. 3A & B). The addition of PGA (0, 31.3, 62.5, 125, 250, or 500 μg/ml) enhanced the cytotoxic effects of LT in a concentration-dependent manner (Fig. 3A & B). The addition of 500 μg/ml PGA to LT containing 0.5 μg/ml PA and 0.1 μg/ml LF or 1.0 μg/ml PA and 0.2 μg/ml LF significantly decreased cell viability by 29.3% (P = 0.00024) and 59.0% (P = 3.235E −08), respectively, compared with samples that did not contain PGA. Treatment with medium alone (control) or 500 μg/ml PGA alone (without LT) was

VL IMGT subgroup

IGKV3-12*01 IGKV3-12*01

VL family

κ3 κ3

JL

2 2

Kd (μM)

0.8 2.6

GenBank accession no. Heavy chain

Light chain

JQ700443 JQ700444

JQ700445 JQ700445

not cytotoxic to BMDMs (Fig. 3A & B). These results confirmed the enhancement effect of PGA on the cytotoxic activity of LT in BMDMs. To evaluate the effect of anti-PGA mAb on PGA-enhanced LT-mediated cell cytotoxic activity, BMDMs were treated with PGA (125 μg/ml) and LT (1.0 μg/ml of PA and 0.2 μg/ml of LF), which pre-incubated with various concentrations of mAb A-12 (3.75, 1.875, 0.937, or 0.468 μg/ml) for 1 h before treatment to BMDMs. After 4-h treatment, cell viability was assessed using MTT assay. As shown in Fig. 3C, treatment with LT alone caused cell death, and the addition of PGA augmented the cytotoxic activity by 33%. These cytotoxic activities were inhibited by the pre-incubation with various concentrations of anti-PGA mAb A-12 with PGA and LT. Pre-incubation with 3.75, 1.875, 0.937, or 0.468 μg/ml of mAb A-12 rescued the cell viability completely (P b 0.05). This result indicates that anti-PGA mAb A-12 inhibits activity of PGA in enhancing LTmediated cell cytotoxic activity probably through specific binding.

3.4. PGA treatment with LT enhances LF activity in mouse BMDMs The most prominent effect of LT on macrophages is the induction of cell death [3,29,30]. The LF present in LT cleaves and inactivates MAPKKs 1, 2, 3, 4, and 6, each of which plays an important role in MAPK-dependent immune activation and inactivation of MAPKKs results in impaired host defenses [3,29,30]. In addition, the recent identification of a susceptibility locus for LT in different mouse strains highlights the role of the inflammasome component, NALP1b, as a potential target of LF in macrophage death [5]. Inflammasome formation involves the association of NALP1 with NOD2 to activate caspase-1, which then triggers the death of host cells through pyroptosis [31]. Caspase-1 is a protease that cleaves the precursor forms of proinflammatory cytokines, such as interleukin (IL)-1β and IL-18, to form active, mature peptides [32]. Caspase-1 itself exists as a proform (p45), and upon activation the proform of caspase-1 is ultimately cleaved into two bioactive forms, p20 and p10 [33]. Previously, we showed that PGA enhances PA binding to J774A.1 cells, resulting in an increased amount of LT inside cells, which then increases the extent of cell death by degrading MAPKK and activating caspase-1 [15]. To confirm these results in mouse BMDMs, lysates from LTand PGA-treated BMDMs were analyzed by western blotting using antibodies that recognize MAPKK 2, 4, and 6 to demonstrate the shutdown of MAPK-dependent signaling pathways through Erk, p38, and JNK. We also used an antibody that recognizes the processed form of caspase-1. As previously reported [15], levels of MAPKK 2, 4, and 6 decreased (Fig. 4A), whereas levels of the cleaved p10 subunit of caspase-1 increased (Fig. 4B) after treatment of BMDMs with LT and increasing concentrations of PGA. The addition of 500 μg/ml PGA significantly decreased the amount of MAPKK 2, 4, and 6 by 77.9%, 86.0%, and 92.8%, respectively (P b 0.05), and increased levels of the cleaved p10 subunit of caspase-1 by 240.7% (P = 0.0017) compared with expression in cells treated with LT only. And pre-incubation of 3.75 μg/ml of mAb A-12 with PGA (125 μg) and LT rescued the PGA activity of enhancing LT-mediated MAPKK6 degradation completely compared to PGA and LT treated control (P = 0.003) (Fig. 4C). In accordance with a previous report [15], our results showed that increased LF activity owing to the presence of PGA led to more death of BMDMs due to increased intracellular LF activity and anti-PGA mAb effectively reversed the effect of PGA.

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whereas 50% of mice that received LT (48 μg PA and 20 μg LF) survived (Fig. 5). The percentage of mice that died rose to 100% (P = 0.00846) for mice that received 500 μg of PGA with addition of LT (Fig. 5). Tail vein injection of PBS (control) did not alter survival rates (Fig. 5). To test whether the anti-PGA mAbs could neutralize PGA in the presence of LT, 0.2 ml PBS containing mAb A-12 (0.1 or 0.5 mg) was injected i.p. 4 h before tail vein injection of 500 μg PGA with LT. IgG was used as a control. Injection of 0.1 mg A-12 rescued the mice by 50% (P = 0.00702), whereas 0.5 mg A-12 rescued the mice completely (P = 0.000045) compared to LT with PGA–treated mice, confirming the effectiveness of anti-PGA mAb (Fig. 5). To further evaluate the functional potency of A-12, 7–9 A/J mice per group were passively immunized i.p. with 0.2 ml A-12 (0.5 or 1 mg). At 4 h after passive immunizations, mice were challenged with a 10 × LD50 dose of B. anthracis H9401 spores by i.p. injection. Injection of 1.0 mg A-12 rescued the mice by 50% (four of eight) compared to PBS-injected control mice (P = 0.025). Injection with 0.5 mg A-12, however, rescued the mice by only 12.5% (one of eight); this was not significantly different from the result obtained for mice injected with negative-control serum (IgG), which protected 11% (one of nine) infected mice (Fig. 6).

3.6. Homology modeling of mAbs and their docking structures with PGA supports specific interactions against PGA antigen

Fig. 1. PGA-specific mAbs A-11 (a–d) or A-12 (e–h) bind to the surface of encapsulated B. anthracis H9401 strain (a & e) but not to the surface of non-encapsulated H9401 lacking pXO2 (c & g). Photomicrographs a, c, e, and g are immunofluorescence images, and b, d, f, and h are phase contrast images.

3.5. mAb A-12 protects mice from PGA-enhanced LT-mediated death and virulent spore challenge Before we tested whether the anti-PGA mAbs could neutralize PGA in the presence of LT, we confirmed the enhancement effect of PGA on LT activity in vivo. Female BALB/c mice (6 weeks old; n = 8) that were given a tail vein injection of 500 μg PGA alone survived,

To further investigate specific interaction between mAbs and PGA, homology modeling of mAbs, A-11 and A-12, was performed and their docking complexes with PGA antigen were analyzed at 2.0 Å resolution. Amino acid sequence analysis revealed that A-11 and A-12 have 89% and 93% sequence similarity with PDB ID: 2UZI which was the most suitable template for homology modeling. Homology modeling by MODELLER 6 provided several structural models for each mAb, which were similar in 3D structures (data not shown). Among these models, Fig. 7A & D show representative superimposition of A-11 and A-12 with the template PDB ID: 2UZI. Final minimized energy levels were −2,252.99 and − 2,510.19 kJ/mol with torsion angle energy of − 23.03 and − 33.76 kJ/mol for A-11 and A-12, respectively. CDR binding site regions of A-11 and A-12 mAbs were identified from alignment with PDB ID: 2UZI. Using these models, interaction of mAb and PGA was analyzed by Dock 6 docking program. For a representative PGA antigen, tri-γ-D-glutamic acid model structure was constructed by Amber program, which resulted in 60 kcal/mol of ΔE after 10 ns molecular dynamics running. After surface analysis and CDR docking site identification, PGA occupied CDR pockets of mAb models and complex models revealed docking features for the antigen PGA (Fig. 7B & E). A-11 complex model suggests that heavy chain CDR2, CDR3, and light chain CDR3 participate in complex formations (Fig. 7C) whereas A-12 complex formation is mainly due to contact of heavy chain CDRs with PGA (Fig. 7F). Detailed inspection of binding pockets of A-11 and A-12 docking model indicates that PGA adapts a position in a cage surrounded by polar and aromatic amino acids or even backbone carbonyl groups (Fig. 8). In A-11 complex shown in Fig. 8A, Arg213 in the light chain CDR3 is critical to PGA binding. Arg213 alone mediates simultaneous two direct hydrogen bonds (1.9 Å and 2.9 Å) and additional salt bridges (3.4 Å) with two carboxy groups of PGA, which stabilize antibody and antigen complex. Leu99 is an additional stabilizer which is located in heavy chain CDR of A-11. A-12 binding site is located mainly in heavy chain CDRs (Fig. 8B). Glu174, Asn102, and Tyr103 are key residues for interaction with PGA antigen. Glu174 makes ionic interaction with alpha amino groups of PGA, whereas Asn102 interacts with alpha carboxy group of PGA. These atomic interactions between mAbs and PGA in both models might support not only micromolar (A-12) and submicromolar (A-11) affinities from SPR experiments but also protective function of mAb against LT with PGA or spore challenge.

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Fig. 2. mAbs A-11 and A-12 show opsonophagocytic activity against B. anthracis H9401. B. anthracis H9401 were incubated with complement, differentiated HL-60 cells, and serial dilutions of A-11 or A-12 mAb fractions. Percentage of killing was calculated relative to growth of bacilli incubated without antibody fraction. Each bar represents the mean ± SD derived from three separate experiments.

4. Discussion The PGA capsule in B. anthracis is first polymerized on the bacterial cell surface to produce a structure of high molecular mass [34]. The capsule is then degraded to a lower molecular mass structure that is released from the cell surface [34]. However, the biological function of PGA in B. anthracis has remained largely unclear, except for its role in protecting bacteria from phagocytosis [1]. Recently, the importance of PGA both in pathogenesis and in prevention and treatment of anthrax has been examined. The PGA capsule can activate caspase-1 and induce the release of IL-1β from THP-1, a human acute monocytic leukemia cells that had differentiated into macrophages and from human monocyte-derived dendritic cells [35]. PGA also increases the cytotoxicity of LT in a concentration-dependent manner in the mouse macrophage cell line J774A.1 by enhancing the binding of PA to its receptor, which results in an increase in intracellular LF. Then, the increased LF causes macrophage death through enhanced degradation of MAPKKs and increased activation of caspase-1 [15]. In accordance with our previous report [15], PGA produced a concentration-dependent enhancement of LT cytotoxicity on mouse BMDMs, and this increase was confirmed using western blotting, which showed that the combination of PGA and LT produced a greater degree of degradation of MAPKKs and an increased level of activation of the proform of caspase-1 to its processed form compared with the effects of LT alone. Our results as well as previous studies [15,35] strongly suggest that the high serum concentrations of PGA observed in later stages of infection with B. anthracis may not only result in septic shock due to an increase in IL-1β but also augment LT cytotoxicity and thereby accelerate toxemia and death of the host [15,35]. Because PGA alone or in combination with LT seems to play an important role in the pathogenesis of anthrax infection, information about concentrations of PGA as well as LT at each stage of B. anthracis infection in various animal models is necessary for understanding anthrax pathogenesis. In the case of rhesus macaques infected with B. anthracis Ames spores by inhalation, PGA in the blood exhibits a triphasic kinetic profile during disease progression [36]. PGA is not detected at 24 h, increases at 48 h, decreases at 72 h, and then increases at 96 h and 120 h. LF also shows a triphasic pattern, whereas PA is not detected until later stages of infection, which may be due to saturation of host receptors, decreased cellular uptake, and thus accumulation of PA in serum [36]. At 96 and 120 h, three of five rhesus macaques showed PA levels ranging from 147 to 19,434 ng/ml [36]. The PGA level in each animal at the terminal stage is much higher than that of PA, ranging from 32,000 to 1,126,388 ng/ml [36]. Bacteremia was also triphasic: it was positive at 48 h, negative at 72 h, and positive at euthanasia [36]. These data

Fig. 3. PGA enhances LT-mediated cytotoxicity in a concentration-dependent manner. The viability of BMDMs was determined after 4-h incubation with LT, which consisted of either 0.5 μg/ml PA and 0.1 μg/ml LF (A) or 1.0 μg/ml PA and 0.2 μg/ml LF (B). PGA was isolated from B. licheniformis ATCC 9945a and added to the LT at concentrations of 0, 31.3, 62.5, 125, 250, and 500 μg/ml before incubation with the BMDMs. After a 4-h incubation, cell viability was determined using the MTT assay. *P b 0.05 vs. LT without PGA control. (C) mAb A-12 inhibits the PGA-enhanced LT-mediated cell cytotoxicity. The viability of BMDMs was determined by the MTT assay after a 4-h incubation with PGA (125 μg/ml) and LT (1.0 μg/ml of PA and 0.2 μg/ml of LF), which were pre-incubated with various concentrations of mAb A-12 (3.75, 1.875, 0.937, or 0.468 μg/ml) for 1 h. *P b 0.05 vs. LT with PGA control. The y axis represents the percent of survival relative to control values, given as the mean ± SD derived from three separate experiments.

indicate an initial rapid and extended clearance of PGA-producing bacilli (72 h) after infection that is followed by a resurgence of bacterial load in the blood of rhesus macaques. In these monkeys, the level of PGA is approximately 100- to 350-fold higher than that of PA at the later stage of infection, suggesting that a high concentration of PGA at this stage may enhance IL-1β production and LT-mediated cytotoxicity and result in accelerated toxemia and cell death [15]. In mice, serum levels of PGA and PA range from 500 to 1,000 μg/ml and 0 to 408 ng/ml, respectively, at later stages of infection, during which the actively produced virulence factors from B. anthracis stimulate cytokine production [37,38]. These reports also support the importance of PGA in anthrax pathogenesis. Previously, we showed that serum containing antibodies against PA, produced by PA vaccination, was effective in both protecting against

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virulent spore challenge and inhibiting the PGA-induced increase in the cytotoxic activity of LT both in vitro and in vivo [15], which supports the use of current PA-based anthrax vaccines. However, the current PA-based vaccine does not elicit the production of anti-capsule

Fig. 5. mAb A-12 provides protection against the PGA-enhanced LT-mediated mortality in BALB/c mice. For the effect of PGA on LT-mediated mortality, groups of mice (6–8 mice per group) were given tail vein injections of PGA (500 μg), LT (48 μg PA and 20 μg LF) or PGA with LT. PBS was used as control. For the protection experiment with anti-PGA mAb, A-12 (0.5 or 1.0 mg) or IgG (1.0 mg) in 0.2 ml PBS was injected into the i.p. cavities of mice 4 h before the tail vein injection of LT (48 μg PA and 20 μg LF) and PGA (500 μg). The survival of mice was monitored daily for 14 days after injection of LT with PGA.

antibodies, and the anti-phagocytic nature of the capsule ensures the unimpeded growth of bacilli. Based on this importance of the PGA capsule in anthrax pathogenesis, efforts toward using PGA as a vaccine component or therapeutic target are progressing. For production of antibodies against PGA, various methods including conjugation to strong immunogenic carriers including PA [16,20] or administration of PGA in combination with a CD40 agonist mAb [18] are used to enhance the weak immunogenicity of PGA. Several murine- and chimpanzee-derived mAbs have been produced and showed effective opsonophagocytosis against B. anthracis [18,19,27]. Passive immunization with these mAbs also confers significant protection in naive mice against spores of the Ames strain [18,19]. Compared to murine mAbs, chimpanzee-derived mAbs show an order of magnitude lower Kd values and confer better protection [27]. Chimpanzee-derived mAbs provide not only pre-exposure protection but also protection against lethal infection when mAbs are administered as late as 20 h after spore challenge [27]. The mAbs produced in our study yielded Kd values similar to those reported for other mouse mAbs. On the other hand, they were 3–4 order of magnitude higher than those for chimpanzeederived mAbs [19,27]. The mouse mAbs that have Kd values of 0.3 μM or 0.5 μM completely protected mice from virulent anthrax spore challenge at 2 mg doses and shows partial protection at 250 μg doses [19], which is similar to our results (Fig. 6). The chimpanzee-derived mAb which has a Kd value of 0.2 nM shows complete protection against spore challenge at a lower dose of antibody (0.3 mg) [27]. Our study also shows that the produced anti-PGA mAb A-12 effectively reversed

Fig. 4. PGA enhances LT activity. Cultures of BMDMs were incubated with LT, consisting of 0.5 μg/ml PA and either 0.075 μg/ml or 0.1 μg/ml LF (for MAPKK and caspase-1 analyses, respectively), and 0, 62.5, 125, 250, or 500 μg/ml PGA for 4 h. Cell lysates derived from the cultures were analyzed with western blotting (30 μg total protein/lane) for the presence of (A) MAPKK2 (upper panel), MAPKK4 (middle panel), and MAPKK6 (lower panel) and (B) for the presence of the activated product of caspase-1, p10 (upper panel). *P b 0.05 vs. LT without PGA control. (C) mAb A-12 inhibits the PGA-enhanced LT-mediated MAPKK6 degradation. The degradation of MAPKK6 was detected by Western blotting after 4-h incubation with PGA (125 μg/ml) and LT, which were pre-incubated with 3.75 μg/ml of mAb A-12 for 1 h. *P b 0.05 vs. LT with PGA control. Western blots were photographed and quantified, and the results are expressed relative to values obtained using α-tubulin as an internal control. Each bar represents the mean ± SD derived from three separate experiments.

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Fig. 6. mAb A-12 protects against anthrax spore challenge in A/J mice. Groups of mice (7–9 mice per group) were injected i.p. with 200 μl IgG (1.0 mg) or mAb A-12 (0.5 or 1.0 mg) in PBS 4 h before 10× LD50 spore challenge by i.p. Control mice were injected with PBS alone. The survival of mice was monitored daily for 14 days.

the enhancement effects of PGA on LT-mediated cytotoxicity (Fig. 3C) and MAPKK6-cleavage (Fig. 4C) in BMDMs. Comparative protein structure modeling is a computational approach to build 3D structural models for proteins using experimental structure of related protein as a template and regular blinding assessment of modeling accuracy showed that comparative protein structure modeling is currently one of most reliable methods to model protein structure [39,40]. Several research groups have reported mAbs with various affinities against B. anthracis capsule [18,19,27], however their molecular interactions with PGA have not been studied. Our results

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(Figs. 7 & 8) provide the first successful 3D models of anti-PGA mAbs at the atomic level using PDB ID: 2UZI as the template, and subsequent antibody-antigen complex analysis gives a more detailed possible feature of interactions between antigen PGA and CDR binding site of mAbs. The binding on A-11 mAb was mainly mediated from simultaneous hydrogen/salt bonds of one critical amino acid Arg213, whereas A-12 complex formation was from concerted contribution of the several amino acids including Glu174, Asn102 and Tyr103 (Fig. 8). Taken together, these molecular interactions at the atomic level might explain Kd value differences from SPR analysis experiment, and in vivo mouse protection against LT with PGA or anthrax spore challenge. Our study with mice is the first to show that mAbs against PGA are effective at inhibiting PGA-mediated enhancement of LT cytotoxicity in addition to their known protective effect against a fully virulent spore challenge primarily owing to the opsonophagocytic activity of the PGA antibodies in combination with the activities of complement and neutrophils. These observations strongly support utilization of PGA antibodies and antibiotics as a clinical treatment in addition to prior immunization with PA-based anthrax vaccines, especially with antibiotic-resistant strains. Further studies are necessary to clarify the relationship between the PGA-neutralizing activity of PGA antibodies and PGA concentration and to find the optimal level of therapeutic anti-PGA antibody for protection against anthrax pathogenesis.

Acknowledgements This work was supported by the Korea National Institute of Health Grant (4840-300-210). We thank Prof. In-san Kim and Dr. Soyoun Kim for providing modelling facility (Kyungpook National Univ.).

Fig. 7. Homology models of A-11 and A-12 and their docking with PGA. A, B & C are modeling results from A-11 mAb whereas d, e & f are from A-12 mAb. Superimposition model from A-11 (A) or A-12 (D) with the template PDB ID: 2UZI, side view of A-11 (B) or A-12 (E) and PGA complex, top view of A-11 (C) or A-12 (F) and PGA complex. In (A) and (D), green lines represent the template light chain while cyan represents the template heavy chain. Yellow and red represent A-11/A-12 light chain and heavy chain, respectively.

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Fig. 8. Binding site residues interacting with bound PGA. PGA binding pocket in A-11 (A) or A-12 (B) mAb. The antigen PGA is surrounded by designated CDR residues. Pink colors highlight PGA backbones. Proposed key binding residues such as Leu99, Arg213 in A-11 and Asn102, Tyr103, Glu174 in A-12 are shown in stick representation.

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