Sequential B-Cell Epitopes of Bacillus anthracis Lethal Factor Bind Lethal Toxin-Neutralizing Antibodies

Mice.Six-week-old A/J strain female mice were purchased from Jackson Laboratories (Bar Harbor, ME). The mice were housed in the Oklahoma Medical Research Foundation Chapman Animal Facility. All mouse experiments were approved by the Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee.

Immunization and blood sampling.For antibody studies, experimental mice were immunized subcutaneously with rLF protein (100 μg) emulsified 1:1 in complete Freund's adjuvant (Difco, Lawrence, KS) on day 0 using a previously described immunization schedule (16). Mice were boosted three times with 50 μg of the immunizing protein emulsified 1:1 in incomplete Freund's adjuvant on days 10, 24, and 38. Control mice were similarly immunized and boosted with adjuvant alone. Retroorbital bleeding of anesthetized mice was performed on days 14, 28, 42, and 116.

Production of rLF protein.rLF protein was produced as an amino-terminal His₆-tagged protein (37, 46). Cultures of BL-21/pET15b (containing LF cDNA from B. anthracis) were grown in Luria broth containing ampicillin (50 μg/ml) to an optical density at 600 nm (OD₆₀₀) of 0.6 to 0.8, and protein expression was induced by addition of 1 mM isopropyl β-d-thiogalactoside (IPTG) and incubation overnight at 16°C. Cells were then pelleted and disrupted with a French press. The lysed cells were then centrifuged, and the supernatant was passed over a Ni²⁺-charged column equilibrated with a binding buffer (Novagen, Gibbstown, NJ). The bound fusion protein was removed with 0.5 M imidazole according to the manufacturer's instructions (Novagen, Gibbstown, NJ). The eluted protein was then dialyzed against 20 mM Tris-HCl (pH 7.5). The protein concentration was determined by a standard Bradford assay (Bio-Rad, Hercules, CA) and stored at 4°C on ice. Purified LF (20 μg) was electrophoresed in a 12.5% sodium dodecyl sulfate-polyacrylamide gel and visualized by Coomassie blue staining to ensure the purity of the sample.

Assessment of LeTx activity.RAW 264.7 mouse macrophages were grown in 96-well plates (1 × 10⁵ cells per well) in 100 μl of Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, 2 mM Glutamax, 2 mM HEPES, and 50 μg/ml gentamicin at 37°C in a 5% CO₂-95% air atmosphere. To assess the functionality of the protein, LeTx was added at a PA-to-LF ratio of 3:1 (75 ng/well PA and 25 ng/well LF; final well volume, 100 μl) and serially diluted 1:2 to assess the point at which toxin activity decreased. Cells treated with medium alone served as negative controls. An additional set of controls involved treatment of cells with LF alone and with PA alone at each concentration. All experiments were done in duplicate. Cells were incubated for 2 h at 37°C, followed by addition of WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (Dojindo Molecular Technologies, Inc., Gaithersburg, MD) at a concentration equivalent to 0.01% of the total volume of the wells. OD₄₅₀ measurements for wells that were subjected to different treatments were analyzed to assess whether LeTx significantly affected RAW 264.7 cell viability.

Standard enzyme-linked immunosorbent assay (ELISA).Ninety-six-well microtiter plates (Corning CoStar, Lowell, MA) were coated with 2.5 μg of antigen/ml in carbonate coating buffer (0.125 μg of antigen/well; pH 9.6) overnight at 4°C. The plates were then washed four times with 0.05% Tween 20 in phosphate-buffered saline (PBS) and blocked with 0.1% bovine serum albumin-0.2% NaN₃ in PBS for 1 h at room temperature. The plates were washed again, and appropriately diluted samples were added and incubated for 2 h at room temperature. After washing, alkaline phosphatase-labeled goat anti-mouse immunoglobulin G (IgG) (Sigma, Saint Louis, MO) diluted 1:5,000 was added. After 2 h of incubation at room temperature, ρ-nitrophenyl phosphate (Sigma, St. Louis, MO) in substrate buffer (0.167 M NaHCO₃, 0.012 M Na₂CO₃, 0.001 M MgCl₂, 0.02% NaN₃) was added to the plates, and the OD₄₁₀ and OD₄₉₀ of the plates were determined.

In vitro LeTx neutralization assay.RAW 264.7 mouse macrophages were grown in Dulbecco's modified Eagle medium containing 4 mM l-glutamine (ATCC) supplemented with 10% fetal calf serum at 37°C in a 5% CO₂-95% air atmosphere. To determine toxin neutralization, 1 × 10⁵ cells were plated in each well of a 96-well flat-bottom tissue culture plate and cultured overnight at 37°C with 5% CO₂. The next day, serum samples were diluted to obtain specific concentrations in culture medium and incubated for 1 h at room temperature with LeTx (composed of PA and LF at a 3:1 ratio [75 ng/well PA and 25 ng/well LF; final well volume, 100 μl]). Following incubation, the medium was removed from the cultured cells, and 100 μl of the serum-toxin mixture was added to each well. Cells treated with medium alone served as negative controls. An additional set of controls involved treatment of cells with LF alone, with PA alone, and with LeTx alone. All experiments were carried out in duplicate. Following addition of the serum-toxin mixture, the cells were incubated for 2 h at 37°C with 5% CO₂. After 2 h, 10 μl of WST-8 (CCK-8; Dojindo Molecular Technologies, Inc., Gaithersburg, MD) was added to each well and incubated for an additional 3 h at 37°C with 5% CO₂. The OD₄₅₀ was determined using a Dynex MRX II microplate reader (Dynex Technologies, Chantilly, VA). The percentage of viability was calculated based on the absorbance for the control wells containing only cells (medium alone).

In vivo LeTx challenge.rLF and PA were produced as described above. rLF-immunized mice and mice treated with adjuvant alone were challenged with 3 A/J 50% lethal doses (LD₅₀) of LeTx (300 μg PA plus 125 μg LF; experimentally determined). Mice were then monitored for 7 days, and mortality was recorded. Survival curves were generated and percentages of survival were determined by using GraphPad Prism (GraphPad Software Inc., La Jolla, CA).

Solid-phase ELISAs.To determine the fine specificity of the anti-LF antibodies, a solid-phase peptide ELISA was used. Peptides that were 10 amino acids long, overlapped by 8 amino acids, and covered the entire length of the LF protein (GenBank accession number AAM26117) were covalently synthesized on polyethylene pins using a 96-well format as previously described (28, 29). Washing and incubation were carried out in sealed plastic containers. Other assay steps were performed by lowering the pins into microtiter plate wells. First, the pins were blocked with 3% low-fat milk in PBS for 1 h at room temperature. The pins were then incubated in 1:200 dilutions of sera in 3% milk-PBS with 0.05% Tween for 2 h at room temperature, washed with PBS-Tween, and incubated with a 1:20,000 dilution of horseradish peroxidase-labeled anti-human IgG conjugate (Kirkegaard & Perry Laboratories [KPL], Gaithersburg, MD) at 4°C overnight. Following washing, the pins were incubated with SureBlue Reserve TMB microwell peroxidase substrate (KPL) for 5 min at room temperature, and the reaction was terminated by addition of TMB stop solution (KPL). The OD₄₅₀ was determined by using a Dynex MRX II microplate reader (Dynex Technologies). The results for each plate were then standardized by comparison with the results for positive control pins. The same control pins were used for all plates and were developed to a specific optical density with a known concentration of a standard control serum. After completion of an assay, the pins were sonicated for 2 h in sonication buffer (40 g sodium dodecyl sulfate, 4 ml β-mercaptoethanol, and 62.4g sodium phosphate in 4 liters) to remove the antibodies, conjugate, and substrate. After sonication, the pins were washed twice in hot water and boiled in methanol for 2 min. The pins were then allowed to air dry for 15 min and were stored with desiccant or used for another assay. An epitope was defined as two or more consecutive solid-phase ELISA pins exhibiting an OD₄₅₀ greater than or equal to the average OD₄₅₀ plus 3 standard deviations (SD) for “adjuvant-only” sera on all decapeptides.

Screening of LF MAbs.LF-specific MAbs were obtained from various sources and used to determine if a neutralizing MAb could bind linear peptides. MAb 10G3 (commercial name, MAb 291) was an antibody that was provided by the Biodefense and Emerging Infections Research Resources Repository (BEI, Manassas, VA). MAb 10G3 was an antibody produced and characterized by Little et al. (27), MAb LF 9A11 was an antibody produced and characterized by Staats et al. (41), and MAb LF8 was an antibody produced and characterized by Zhao et al. (51). All antibodies were screened as described above for rLF-specific polyclonal antisera.

Assay for in vitro peptide inhibition of neutralization.Peptides (10-mers) (see Fig. 4B) bound by MAbs were synthesized as linear peptides by using standard Fmoc chemistry and were purified by high-performance liquid chromatography to obtain ≥95% purity (GenScript, Piscataway, NJ). Peptide inhibition of MAb-mediated toxin neutralization was achieved by titrating various concentrations of peptides into tubes containing the corresponding MAbs at fixed concentrations. The peptide-antibody mixtures were incubated for 30 min at room temperature. After incubation, LeTx (at the concentration used in the in vitro neutralization assay described above) was added to each tube and incubated for 30 min at room temperature. The concentration of each MAb was chosen so that it was the last 2× dilution exhibiting maximal neutralization capacity with a starting MAb concentration of 5 μg/ml. Following incubation, the medium was removed from the cultured cells, and 100 μl of the peptide-antibody-toxin mixture was added to each well. Cells treated with medium alone served as negative controls. All experiments were carried out in duplicate. Following addition of the peptide-antibody-toxin mixture, the cells were incubated for 2 h at 37°C with 5% CO₂. After 2 h, 10 μl of WST-8 (CCK-8; Dojindo Molecular Technologies, Inc., Gaithersburg, MD) was added to each well and incubated for an additional 3 h at 37°C with 5% CO₂. The OD₄₅₀ was determined using a Dynex MRX II microplate reader (Dynex Technologies, Chantilly, VA). The percentage of viability was calculated based on the absorbance of the control wells containing a mixture of irrelevant control peptide, antibody, and toxin.

rLF immunization results in high titers of neutralizing anti-LF antibodies and protection from LeTx challenge.Groups of A/J mice were immunized on day 0 with rLF in complete Freund's adjuvant and boosted with rLF in incomplete Freund's adjuvant on days 10, 24, and 38. Sera were collected 3 to 4 days after each boost and again at day 116 to determine the IgG anti-LF antibody levels by ELISA. The antibody titers were high by 2 weeks after the initial immunization and reached maximum levels (>1 × 10⁶) by day 28 (Fig. 1A). The titers of anti-rLF IgG were relatively undiminished at day 116, nearly 80 days after the final booster immunization. Sera collected from mice immunized with adjuvant alone (control group) failed to bind LF antigen, as expected.

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FIG. 1.

Immunization of A/J mice results in high-titer anti-LF antibodies that are neutralizing in vitro and protective in vivo. A/J mice were immunized with either rLF or adjuvant alone on days 10, 24, and 38 and bled on days 14, 28, 42, and 116. (A) IgG anti-LF antibody titers of pooled sera from A/J mice immunized with rLF or adjuvant alone were assessed at specified time points to quantitate the IgG anti-LF response. Antibody levels were measured by using a standard ELISA, OD₄₁₀, and OD₄₉₀. (B) Sera collected were subjected to an in vitro neutralization assay using a 3:1 ratio of PA to LF (LeTx). Sera exhibited near-maximal neutralization of toxin activity at day 116 (78 days after the final booster immunization). (C) A/J mice immunized with rLF are protected from an in vivo LeTx challenge using 3 times the A/J LD₅₀ derived experimentally (300 μg PA plus 125 μg LF) for LeTx (P = 0.009).

To determine whether the antisera of rLF-immunized A/J mice contained LeTx-neutralizing activity, serum samples obtained at various time points were assessed for the capacity to neutralize LeTx in vitro in a RAW 264.7 mouse macrophage cell death assay. Various dilutions of antisera were preincubated with a LeTx concentration (3:1 molar ratio of PA to LF) previously determined to result in maximal cell death and then added to RAW 264.7 mouse macrophage cultures. Measurement of macrophage cell viability at 24 h revealed the presence of significant neutralizing antibodies at day 14 (75% viability at a 1:300 serum dilution) and maximal titers of neutralizing antibodies as late as day 116 (100% protection at a 1:900 dilution) (Fig. 1B). Sera from mice immunized with adjuvant alone exhibited no capacity to neutralize LeTx activity.

Groups of rLF-immunized mice and controls immunized using the same schedule but with PBS-adjuvant alone were then challenged in an in vivo setting with three times the experimentally derived A/J LD₅₀ for LeTx (300 μg PA and 125 μg LF) (data not shown) to determine whether the anti-LF responses raised could protect mice from LeTx in vivo. As shown in Fig. 1C, seven of eight A/J mice immunized with rLF were protected from LeTx challenge, while only one of seven A/J controls immunized with adjuvant alone survived the challenge (P = 0.009). These findings reveal that rLF induces a robust immune response that results in neutralization of toxin and protection both in vitro and in vivo, further supporting previously described data (1, 9, 23, 27, 41, 51) indicating that LF can provide protection against a LeTx challenge.

Sequential mouse B-cell epitopes cluster in PA and substrate-binding domains of LF.To analyze the fine specificity of the neutralizing antisera from rLF-immunized A/J mice, we subjected their sera to solid-phase peptide mapping utilizing a modified ELISA performed with a series of decapeptides that overlapped by eight amino acids and represented the whole protein. Reactive epitopes were defined by carrying out three independent immunization and mapping experiments. Figure 2 shows the mapping results for LF-immunized A/J mice for day 14, 28, and 42 bleeds compared to the day 42 results for mice immunized with adjuvant alone (control group). A total of 16 major antigenic regions throughout the molecule were identified. All 16 epitopes defined at day 42 were still present at day 116, although the intensities of epitopes 6, 10, 11, and 15 had increased by that time (data not shown). Mice immunized with adjuvant alone did not significantly bind any of the peptides. An antigenic epitope was defined as described in Materials and Methods. These epitopes were ranked in order of reproducibility and reactivity based on the results of three separate immunization and mapping experiments. The reproducibility and optical density (average ± standard error of the mean) are shown for each defined epitope in Table 1.

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FIG. 2.

Fine specificity of the humoral anti-LF response following A/J immunization. IgG antibody binding to decapeptides of LF overlapping by eight amino acids was measured using a solid-phase ELISA. The data are the antibody responses in sera from rLF-adjuvant-immunized mice on days 14, 28, and 42 and in sera from mice immunized with adjuvant alone. The data are the averages of the results of three independent immunization and mapping experiments. Mapping within each experiment was performed using sera pooled from 18 to 20 mice. Epitopes were numbered in order of reproducibility and reactivity at day 42, as shown in Table 1. The epitope numbers correspond to the epitope numbers in Tables 1 and 2. The horizontal lines indicate extended antigenic regions of epitopes 3 and 9 as defined at day 42.

The 16 identified epitopes shown in Fig. 2 are superimposed on the LF crystal structure in Fig. 3. Sequences, domain locations, and proposed functions of the epitopes are shown in Table 2. Interestingly, of the 16 epitopes identified by this method, the most reproducible epitopes tended to cluster in domains I (PA-binding domain) and III (substrate recognition domain, which has some residues that contact the mitogen-activated protein kinase kinase [or MEK] substrate) (Fig. 3 and Table 2). Specifically, five epitopes were in the PA-binding domain, one epitope overlapped the PA-binding domain and the VIP2-like domain, four epitopes were in the substrate recognition domain, two epitopes were in the VIP2-like domain, and four epitopes were in the zinc-binding domain (Table 2).

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FIG. 3.

Sequential epitopes of LF. The 16 most reactive A/J epitopes are superimposed on the LF crystal structure (33) (Protein Data Bank accession code 1J7N [7]). Roman numerals indicate structural domains. The epitope numbers correspond to those shown in Fig. 2 and Tables 1 and 2.

Two of the five most reactive and reproducible epitopes bound antigenic regions in the PA-binding domain. The PA-binding domain has been shown to bind LF-neutralizing antibodies, directly inhibiting the binding of LF to PA to neutralize LeTx (9, 27, 34, 50). Three of the five most reactive and reproducible epitopes, which were identified in all three independent mapping studies and had average optical densities ranging from 0.75 to 0.8, were located in the substrate-binding region. This domain, identified as a 101-residue segment containing five tandem repeats, was previously shown to be required for LF activity based on insertion mutagenesis (33) and to bind neutralizing antibodies (26, 33, 51); however, the fine specificities of the MAbs were not reported previously.

LF-specific neutralizing MAbs bind epitopes identified in LF-immunized mice.To determine whether sequential epitopes of LF identified in anti-LF antisera could bind neutralizing antibodies, we screened a panel of known LeTx-neutralizing MAbs with specificity for LF using solid-phase epitope mapping and in vitro neutralization assays (27, 41, 51). As shown in Fig. 4A, the neutralizing capacity of all three anti-LF MAbs tested (MAbs 10G3, 9A11, and LF8) was verified, although there was with some variability across the MAbs. MAb 9A11 had the greatest neutralization capacity (93.5% at concentrations as low as 0.313 μg/ml), while MAbs 10G3 (92% at a concentration of 5 μg/ml) and LF8 (80% at a concentration of 5 μg/ml) also exhibited high neutralizing activity.

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FIG. 4.

Neutralizing LF-specific MAbs bind sequential B-cell epitopes of LF. (A) Anti-LF MAbs 10G3, 9A11, and LF8 (27, 41, 51) neutralize anthrax LeTx in vitro. (B) Epitope mapping of LF-specific, LeTx-neutralizing MAbs 10G3, 9A11, and LF8. The sequences of bound decapeptides, the numbers of SD above the background level, and the corresponding epitopes shown in Table 1 are indicated. The colored letters correspond to the colored letters in panel C. (C) Structural representation of five epitopes bound by neutralizing LF-specific MAbs. The colored letters correspond to the colored letters in panel B. Two residues of epitope c are not resolved in the LF crystal structure and thus are not shown. (D) Linear peptides corresponding to MAbs 10G3, 9A11, and LF8 mediate measurable reductions in neutralization in vitro (striped bars). Inhibition of neutralization is compared to the results for the same MAb mixed with toxin and irrelevant peptide from ovalbumin (OVA) (open bars). All experiments were carried out in duplicate. Since duplicate values were nearly identical, error bars are not shown. The concentrations of MAbs used for peptide inhibition (see Materials and Methods) were 1.25 μg/ml for MAb 9A11 and 5 μg/ml for MAbs 10G3 and LF8.

To examine the fine specificity of these neutralizing LF MAbs, we performed a standard solid-phase peptide assay as described above. As shown in Fig. 4B, all MAbs bound linear epitopes of LF to various degrees. Furthermore, the decapeptides bound by these MAbs either coincided with or overlapped epitopes identified by using polyclonal anti-LF antisera (Fig. 2 and Table 1). MAb 10G3 bound an epitope that overlapped epitope 4, located in the substrate recognition domain, at a level 52 SD above the background level. The sequence (DIRDSLSEEE) overlapped epitope 4 (SEEEKELLNRIQ) by four amino acids. MAb 9A11 bound two different epitopes, an epitope that coincided with epitope 13 (domain IV) at a level 9 SD above the background level (INDQIKFIIN) and an epitope not identified by using polyclonal anti-LF antisera at a level 8 SD above the background level (VTNYLVDGNG). Lastly, MAb LF8 also bound two sequences that exhibit significant sequence similarity, one that strongly bound and coincided with epitope 2 (EEKELLKRIQ) at a level 379 SD above the background level and one that coincided with epitope 13 (TEEKEFLKKL) at a level 36 SD above the background level. These epitopes have the sequence EEKEXLK (R/K)(I/L), where X indicates a position where there is no amino acid similarity, and both occur in the substrate recognition domain. These data indicate that linear peptides of LF identified in our study bind neutralizing LF MAb.

To determine whether the peptide sequences of these epitopes had the capacity to inhibit neutralization of the corresponding MAbs, linear peptides corresponding to epitopes 2, 4, and 13 and the VTNYLVDGNG sequence were synthesized. These peptides were used in a modified in vitro neutralization assay to verify the binding specificity of MAbs and their ability to neutralize LeTx. As shown in Fig. 4D, all peptides mediated measurable reductions in MAb-mediated toxin neutralization activities in dose-dependent fashions. Two of these peptides (epitopes 2 and 4) essentially completely blocked neutralization under the conditions tested, whereas peptides corresponding to both epitopes recognized by MAb 9A11 exhibited measurable but less potent inhibition of MAb 9A11 neutralization capacity. Inhibition of neutralization was determined by comparing the percentages of viability of macrophages exposed to mixtures of MAb, toxin, and peptide to those of mixtures of MAb, toxin, and irrelevant control peptide from ovalbumin (ISQAVHAAHAEINEAGR) (control). The data obtained further supported the binding of linear peptides by LeTx-neutralizing antibodies. To our knowledge, this is the first time that linear peptides have been shown to inhibit neutralization of LeTx by these MAbs (27, 41, 51).