PA was prepared from B. anthracis
as previously described (33
). EF was isolated from an Escherichia coli
expression system and was purified as previously described (29
). The chimpanzee anti-EF IgG EF13D was produced as previously described (4
Calmodulin was expressed in E. coli
as previously described. Briefly, BL21(DE3) Gold cells harboring the plasmids pProEx-modified-rCaM and pUBS520 (kind gifts of Wei-Jin Tang, University of Chicago) (6
) were grown in autoinducing medium ZYM-5052 (30
) for 24 h at 30°C while shaking. Centrifugation, cell lysis, and purification using phenyl-Sepharose were accomplished as detailed by Maune and colleagues (20
EF truncation mutant plasmids were described previously (4
). These plasmids use a pET31b backbone and harbor the various EF truncations shown in , with a C-terminal His6
tag (GenBank accession numbers JN540812
). Plasmids were transformed into E. coli
strain BL21(DE3) Gold. Cells were grown overnight in Terrific broth (12 g tryptone, 24 g yeast extract, and 4 ml glycerol per liter, buffered with 17 mM KH2
and 72 mM K2
) with 100 μg/ml ampicillin at 37°C, then subcultured 1:10 into the same medium at 37°C for 4 h, centrifuged, and resuspended in 1× IMAC buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 10 mM imidazole, 0.1% Tween 20). Cells were lysed by ultrasonication, cell debris was pelleted, and the supernatant was incubated with Ni-nitrilotriacetic acid (NTA) agarose beads (Qiagen, Germantown, MD) on ice for 1 h with gentle shaking. After thorough washing, the proteins were eluted with 250 mM imidazole in 1× IMAC buffer. Proteins were dialyzed into HBS-T (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Tween 20) by repeated washing in an Amicon Ultra diafiltration cartridge with a 5,000-molecular-weight-cutoff membrane (Millipore, Billerica, MA).
Mouse immunization, hybridoma fusion, antibody expression, purification, and subtyping.
Two BALB/cJ mice that were initially 10 weeks old were immunized subcutaneously (s.c.) with EF (10 μg/100 μl in phosphate-buffered saline [PBS], without adjuvant) five times, at 2-week intervals, followed by a sixth and final immunization 4 weeks after the fifth immunization. Mice were bled to assess the immune response and spleens collected 3 days after the final boost. Splenocytes isolated from the EF-immunized mice were fused to SP2 cells (ATCC, Manassas, VA) by standard methods to produce hybridoma clones that were grown in 96-well plates in HAT medium (RPMI 1640 supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100 μg/ml penicillin-streptomycin, 10 μg/ml gentamicin, all from Invitrogen, Carlsbad, CA, further supplemented with 100 μM hypoxanthine, 0.4 μM aminopterin, and 800 μM thymidine [Sigma]). Supernatants were collected after 7 to 14 days and stored at −80°C until used for the initial enzyme-linked immunosorbent assay (ELISA) screen. Positive clones were plated in HT medium (HAT medium lacking aminopterin [Sigma]) in 24-well plates, subcloned, expanded, and frozen in 10% dimethyl sulfoxide (DMSO)–90% fetal bovine serum (FBS).
Scale-up of antibody expression was accomplished by culturing hybridoma cell lines in Cell Stack culture chambers with CellBind surface treatment (Corning). Hybridomas were cultured in RPMI 1640 with 10% FBS supplemented with 2 mM Glutamax (Invitrogen), 1 mM sodium pyruvate, and 10 μg/ml gentamicin. Medium was harvested, sterile filtered, and stored at 4°C until purification. Purification was carried out using a Protein A HiTrap MabSelect SuRe column (GE Healthcare, Piscataway, NJ) under high salt conditions on an ÄKTA FPLC (GE Healthcare) as detailed by Harlow and Lane (12
). The fractions containing antibody were pooled and dialyzed against a buffer of 10 mM HEPES, 300 mM NaCl, and 0.5 mM EDTA. Concentrations were assessed by measuring the A280
on an Ultrospec 4000 UV/visible spectrophotometer (GE Healthcare), where 1 mg/ml was equated to an A280
of 1.4. Purity was assessed by nonreducing SDS-PAGE, and activity was confirmed by mixing of antibody with EF followed by resolution using native PAGE to observe a supershift attributed to complex formation. Antibodies were stored at −80°C. Subtyping of antibodies was performed using a rapid isotyping kit for mice (Thermo Scientific, Rockford, IL).
Initial ELISA screen.
Enzyme immunoassay (EIA) 96-well plates (product no. 3590; Corning, NY) were coated with EF (10 μg/ml, or 111 nM) in PBS (5.6 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 154 mM NaCl) overnight at 4°C. After the plates were washed twice with PBS, the hybridoma supernatants were mixed 1:1 with 10 mM Tris, pH 8.0, 100 mM NaCl containing either 2 mM EDTA or 0.5 mM EDTA, 2 mM CaCl2, and 10 μg/ml (0.6 μM) calmodulin (alternate incubation buffers) and incubated for 1 h at room temperature. After five washes with the incubation buffer, anti-mouse antibody conjugated to alkaline phosphatase (1:2,000 dilution; Southern Biotech, Birmingham, AL) prepared in the previous buffer was added for 1 h. Plates were washed five times, and then detection was accomplished with a p-nitrophenyl phosphate liquid substrate system (Sigma). The reaction was stopped with 3 M NaOH upon saturation of signal of at least one clone, and plates were read at 405 nm with a 96-well plate spectrophotometer. Previously verified mouse polyclonal antiserum to EF was used as a positive control, while the lack of primary antibody was the negative control. An absorbance of 0.1 or greater was considered a positive signal.
Candidate monoclonal antibodies (MAb) were then screened by surface plasmon resonance (SPR) for binding to EF by using a ProteOn XPR36 (Bio-Rad, Hercules, CA). EF was immobilized onto a general layer compact (GLC) low-binding-capacity chip by using standard amine-coupling procedures. Briefly, a GLC chip was activated with 0.4 M ethyl(dimethylaminopropyl) carbodiimide (EDC)–0.1 M N-hydroxysulfosuccinimide (sulfo-NHS) at a 30 μl/min flow rate for 300 s. EF was immobilized by passing a 10 μg/ml (111 nM) solution in 10 mM acetate buffer at pH 5.5 for 60 s at a 25 μl/min flow rate. The surface was deactivated with an injection of 1 M ethanolamine at 30 μl/min for 300 s. This resulted in approximately 960 resonance units of immobilized EF. Human serum albumin (Sigma) was similarly immobilized as a negative control, and a surface that was not activated acted as an additional negative control. Undiluted supernatants from each clone were injected as the analyte at 25 μl/min for 60 s, and dissociation was monitored for 600 s. Regeneration was accomplished with a 30 s injection of 4 M MgCl2 at 50 μl/min.
Kinetic parameters of the purified antibodies were determined using the ProteOn XPR36. Each antibody was immobilized on a GLC chip in 10 mM acetate buffer, pH 5, by using standard amine-coupling conditions. This resulted in approximately 961 to 1,225 resonance units of immobilized antibody. A concentration series of EF from 75 nM to 18.75 nM was injected at 100 μl/min for 60 s, and dissociation was monitored for 10 min. Regeneration was accomplished by a 30-s injection of 100 mM HCl at 100 μl/min. Samples were run in triplicate. Data were analyzed by using ProteOn Manager software with the Langmuir model.
Epitope mapping was accomplished using the same surface as for kinetic analysis. A 22 nM injection of EF at 100 μl/min for 60 s was immediately followed by a 15 μg/ml (100 nM) IgG injection at 100 μl/min for 60 s. Because of the multiplex nature of the ProteOn system, all antibodies were immobilized in their vertical lanes, and EF followed by different IgGs was injected in the horizontal direction. In this way, the entire matrix of antibody epitopes could be analyzed with respect to one another in a single experiment. Regeneration was performed as previously noted.
Epitope mapping by EF truncation ELISA.
ELISAs were used to identify the domains of EF to which each antibody binds. EIA plates were coated with the purified anti-EF MAb at 2.1 μg/ml (14 nM) in PBS overnight at 4°C. The plates were washed with PBS-T (PBS with 0.05% Tween 20) and blocked with 2% milk (Bio-Rad) in PBS-T for 3 h. The plates were then washed with PBS-T, and the EF truncation variants were allowed to incubate for 1 h at room temperature in 2% milk–PBS-T. After washing in PBS-T, anti-penta-His horseradish peroxidase (HRP) at a 1:1,000 dilution (Qiagen) was allowed to bind for 1 h at room temperature in 2% milk–PBS-T. After washing, detection was accomplished with 3,3′,5,5′-tetramethylbenzidine substrate (TMB; R&D Systems, Minneapolis, MN), quenched with 1 M H2SO4, and absorbance was read at 405 nm.
EF neutralization in tissue culture.
RAW 264.7 macrophage cells were grown in Dulbecco's modified Eagle's medium (DMEM) with Glutamax (Invitrogen), supplemented with 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES buffer, pH 7.3, and 10 μg/ml gentamicin. Cells were plated at 80 to 90% confluence 24 h prior to toxin treatment. PA (500 ng/ml, or 6 nM), EF (2.25 μg/ml, or 25 nM), and 500 μM 3-isobutyl 1-methylxanthine (IBMX; A. G. Scientific, San Diego, CA) combined in culture medium were incubated with various concentrations of IgG at room temperature for 30 min prior to addition to cells. Cell culture medium was aspirated from the plates, and the toxin-antibody mix was placed on cells for 1 h at 37°C, then cells were lysed with Cisbio lysis buffer supplemented with 50 mM EDTA (to stop enzyme activity), and cAMP levels were assessed according to manufacturer's protocol by using the cAMP HiRange HTRF kit (Cisbio, Bedford, MA).
Western blot analysis.
Purified proteins (50 ng each PA, LF, or EF) were run on Novex 4 to 20% Tris-glycine gradient gels (Invitrogen) under denaturing conditions and then transferred to nitrocellulose membranes, which were then blocked for 30 min at room temperature in 4% milk in PBS-T. Each separate membrane was then exposed to a different monoclonal antibody or the control mix of polyclonal antisera to assess specificity of the primary antibody. MAb to EF were incubated with the membranes at a concentration of 1.4 μg/ml (9.5 nM). PA, LF, and EF controls were detected with polyclonal rabbit antisera developed in our laboratory and used at 1:1,000 dilutions. All antibodies were incubated with the blots in 4% milk–PBS-T overnight at 4°C. After washing in PBS-T, secondary antibodies (goat anti-mouse IgG–IRDye700DX and goat anti-rabbit IgG–IRDye700DX; Rockland, Inc.) were applied at 1:5,000 dilutions in 4% milk–PBS-T for 1 h. After washing in PBS-T, membranes were imaged with a Licor Odyssey (Lincoln, NE) using infrared fluorescence detection. SeeBlue Plus2 prestained standard was the molecular weight marker used (Invitrogen).
EF catalytic activity capture assay.
EIA 96-well plates were coated with anti-EF antibodies at 2.1 μg/ml (14 nM) in HBS (10 mM HEPES, pH 7.4, 150 mM NaCl) overnight at 4°C. Plates were then washed and blocked at room temperature for 3 h in 2% milk in PBS-T. A series of EF concentrations were allowed to incubate for 1 h at room temperature in 2% milk–PBS-T. After washing in PBS-T, 100 μl of 20 mM MnCl2
, 80 μg/ml bovine serum albumin (BSA; 1.2 μM), 20 μg/ml calmodulin (1.2 μM), 1 mM ATP, and 10 mM HEPES, pH 7.3, was added and allowed to incubate for 1 h at 37°C to allow EF synthesis of cAMP (reaction conditions were chosen based upon several previous studies) (14
). Then, 100 μl of Cisbio lysis buffer supplemented with 50 mM EDTA was added and cAMP levels were assessed by using the cAMP HiRange kit (Cisbio).
Spores were prepared from Ames 35, the avirulent, nonencapsulated, toxigenic B. anthracis
), as previously described (13
). Briefly, bacteria were grown at 37°C overnight followed by 7 days at 28°C on NBY sporulation agar (8 g nutrient broth, 3 g yeast extract, 15 g agar per liter water) and were then monitored by microscopy to confirm that sporulation was greater than 95%. Spore purification was accomplished through four cycles of centrifugation followed by washing in sterile water, and spores were then subjected to heat treatment at 70°C for 0.5 h. Spore quantification was carried out using a Petroff-Hausser counting chamber (Hausser Scientific, Horsham, PA) and verified by dilution plating.
In vivo studies.
Female BALB/cJ or C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were used at 8 to 12 weeks of age. For the footpad edema model, BALB/cJ mice (6 to 9/group) were injected in one footpad (20 μl) with ET (0.5 μg EF plus 0.5 μg PA) premixed with either antibody (3.6 μg) or PBS. Footpad edema was monitored 24 h after injection by measuring footpads in the dorsal/plantar direction by using digital calipers (Mitutoyo Corporation, Aurora, IL). In experiments assessing leg edema following spore infection, C57BL/6J mice (4/group) were injected intravenously (i.v.) with 71.4 μg of antibody prepared in 100 μl PBS. After a delay of 10 min, the mice were infected with 2 × 107 spores (20 μl) via s.c. injection in the right foreleg. The limited edema spread in this location allows consistent quantification of edema. Edema was assessed at 24 h after infection by measuring the limb in the sagittal dorsal/ventral direction by using digital calipers. To test antibody efficacy against ET lethality, C57BL/6J mice (5/group) received a single antibody injection (10.7 to 35.7 μg, i.v.) 1 h prior to administration of a lethal dose of ET (25 μg EF plus 25 μg PA, i.v.) and survival was monitored for 200 h. To test antibody efficacy against spore infection, C57BL/6J mice were injected with a cocktail of all three antibodies (7F10, 3F2, and 4A6, 12.5 μg each, i.v., n = 5) or with EF13D (25 μg, i.v., n = 8) at 18 h and 1 h prior to administration of a lethal dose of spores in the scruff of the neck (2 × 107, s.c.). Survival was compared to that of control mice, which received PBS instead of antibody (n = 18). All mouse experiments were performed under protocols approved by the Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.