An Anthrax Lethal Factor-Neutralizing Monoclonal Antibody Protects Rats before and after Challenge with Anthrax Toxin
http://www.100md.com
感染与免疫杂志 2005年第10期
R&D Center, Aprogen, Inc., Bio Venture Center #311, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejon 305-333, South Korea
Laboratory of Antibody Engineering, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejon 305-333, South Korea
ABSTRACT
Lethal factor (LF) is a component of anthrax lethal toxin (LeTx). We generated anti-LF murine monoclonal antibodies (MAbs) that show LeTx-neutralizing activity in vitro and in vivo. Anti-LF MAbs were generated by immunization with recombinant LF, and the MAbs showing LeTx-neutralizing activity in vitro were selected. Two MAbs with the highest affinities, 5B13B1 (dissociation constant [Kd], 2.62 nM) and 3C16C3 (Kd, 8.18 nM), were shown to recognize the same or closely overlapping epitopes on domain III of LF. The 50% inhibitory concentration of 5B13B1 (0.21 μg/ml) was approximately one-third that of 3C16C3 (0.63 μg/ml) in the in vitro LeTx-neutralization assay. The 5B13B1 antibody, which had the highest neutralizing activity, provided perfect protection against LeTx challenge in an in vivo LeTx neutralization assay using Fisher 344 rats. In addition, the antibody showed pre- and postexposure prophylactic effects in the animal experiments. This is the first report that an MAb binding to domain III of LF has neutralizing activity against LeTx. The 5B13B1 antibody may be useful in prophylaxis against anthrax poisoning.
INTRODUCTION
Bacillus anthracis, the causative agent of anthrax, is a spore-forming, gram-positive bacterium. Infection by inhalation of the spores of B. anthracis can result in a mortality rate of up to 80% if the infection is left untreated. This organism was one of the first biological warfare agents to be developed and continues to be a major threat in this regard (8, 14, 25). Anthrax is delivered as inert spores, which germinate into replicating bacteria that produce anthrax toxins. During a systemic infection, B. anthracis can replicate to produce high levels in the bloodstream. At this point in the infection, although aggressive antibiotic therapy can prevent bacterial growth, infected individuals can still die, most probably as a result of high concentrations of bacterial toxins already accumulated in the body (21). Thus, an ideal counterattack should include both killing the B. anthracis with antibiotics and neutralizing the toxin (5). In fact, a combination of antibiotic and immunoglobulin therapy was shown to be more effective than antibiotic treatment alone in a rodent anthrax model (9).
Anthrax toxin consists of three components, designated protective antigen (PA), lethal factor (LF), and edema factor (EF), that together form a tripartite protein exotoxin (15). LF along with PA forms a toxin referred to as lethal toxin (LeTx). After binding to the cell surface receptors TEM8 or CMG2, PA is cleaved into two fragments by a furin-like protease. This allows the carboxy-terminal fragment, PA63, to heptamerize and bind LF or EF. The resulting complexes of (PA63)7 with LF or EF are taken up into cells by receptor-mediated endocytosis and move to an endosomal compartment. The translocation of bound LF or EF into the cytosol is promoted by the structural change of PA induced by an acidic environment (3, 23).
LF is the major virulence factor and is responsible for shock and death (7, 18-20). LF is a zinc-dependent protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near their amino termini. This leads to the inhibition of one or more signaling pathways and thus causes lysis of macrophages (17, 24). LF also offers B. anthracis an efficient mechanism to evade the innate immune response by inhibiting interferon regulatory factor 3 activation by lipopolysaccharide and subsequent cytokine production through bacterial membrane components. In addition, LF severely impairs the function of dendritic cells by disrupting the mitogen-activated kinase intracellular signaling network (1, 6).
Some studies have shown that passive transfer of a neutralizing polyclonal antibody or monoclonal antibody (MAb) can protect cells against anthrax toxin or bacterial challenge (2, 4, 10, 12, 13, 22, 24). The protective efficacy of neutralizing antibody was greatly enhanced by a combination of a PA-neutralizing antibody and an LF-neutralizing antibody (4). Nevertheless, PA has been the primary target for passive protection, as the current approved anthrax vaccine consists principally of PA. In contrast, only a couple of LF-neutralizing MAbs have been described (13, 24). These MAbs were shown to interfere with the binding of LF to PA, but neither their epitope specificities nor the neutralization mechanism was studied.
In this study, we generated LF-neutralizing MAbs that specifically bind domain III of LF, and we confirmed their protective efficacy by performing in vitro and in vivo LeTx neutralization assays. One of the MAbs (5B13B1) protected Fisher 344 rats from LeTx challenge when it was administered before or after exposure to LeTx. The mechanism of neutralization by this MAb is discussed below.
MATERIALS AND METHODS
Expression and purification of PA and LF. The DNA encoding PA was prepared by digestion of pT7-PA (kindly provided by W. K. Seong at the Korea Center for Disease Control and Prevention) with BamHI and SalI and was ligated into the BamHI-SalI sites of pBS1-1 (Aprogen, Korea), which contains an S1 tag at the 5' end of the fused gene (16), to construct pBS1-1-PA. The DNA encoding LF was synthesized by PCR from pXO1 DNA (kindly provided by W. K. Seong. Sung at the Korea Center for Disease Control and Prevention) using a 5' primer (5'-CGTGGATCCATGGCGGGCGGTCATGGTGATG-3') and a 3' primer (5'-GATTCTAGATTATGAGTTAATAATGAAC-3'). The PCR products were digested with BamHI and XbaI and ligated into the BamHI-XbaI sites of pBS1-1 to construct pBS1-1-LF.
To express PA and LF in bacteria, each of the expression plasmids was introduced into Escherichia coli HB2151, and the fresh transformants were grown in 2x YT medium supplemented with ampicillin (100 μg/ml) at 37°C. Induction of gene expression by addition of 1 mM isopropyl--D-thiogalactopyranoside (IPTG) was performed at 30°C for 4 h. The proteins were purified from cleared bacterial lysates by affinity chromatography on an AP1 (murine anti-S1-tagged MAb)-conjugated Sepharose column (Aprogen, Korea). To remove the S1 tag from the S1-tagged LF (S1-LF) and PA (S1-PA), purified S1-LF and S1-PA were incubated with thrombin (1 U/100 μg of fusion protein) at room temperature for 2 h. The fractions containing the toxin protein were separated by fast protein liquid chromatography on a Superose 6 column (Amersham). The purity of the purified proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Generation of LF-neutralizing MAbs. BALB/c mice were immunized with the purified LF by subcutaneous injections. For the first injection, 20 μg of the immunogen was emulsified with complete Freund's adjuvant (Sigma). Three subcutaneous booster injections of LF in incomplete Freund's adjuvant (Sigma) were given every 2 weeks. Fourteen days later, the mice received the last intravenous injection without adjuvant. Splenocytes were collected 3 days later and were immortalized by fusion with mouse myeloma cells (F0; ATCC CRL-1645). The cells were grown in 96-well plates in HAT medium, and the culture medium was screened by an enzyme-linked immunosorbent assay (ELISA) using the purified LF as a coating antigen. The positive clones were screened for LeTx-neutralizing activity by an in vitro macrophage lysis assay. Hybridomas showing LF-binding activity and LeTx-neutralizing activity were subcloned by limiting dilution. MAb was purified from the culture supernatant of hybridomas grown in serum-free medium by affinity chromatography on a protein G-Sepharose column (Amersham).
ELISA. To detect the antibody secreted by hybridomas, each well was coated with the purified LF (2 μg/ml). Then 100 μl of the culture supernatant was added to each well. After three washes, 50 μl of horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (IgG) (Fc-specific) antibody (Pierce) was added to each well and incubated for 1 h at 37°C. After washing, 0.2 M citrate-PO4 buffer (pH 5.0) containing 0.04% ortho-phenylenediamine dihydrochloride and 0.012% H2O2 was added to each well. The reaction was stopped by adding 2.5 M H2SO4, and the absorbance was determined at 492 nm using an ELISA reader (SOFTmaxPRO; Molecular Devices, United States).
For the competition binding assay, each well was coated with the purified LF (100 ng) and blocked with 2% bovine serum albumin for 1 h at 37°C. After washing, biotinylated 5B13B1 and serially diluted unlabeled 5B13B1 or 3C16C3 were added to each well, and the plate was incubated at 37°C for 1 h. After washing, streptavidin-HRP (Pierce) was added for 1 h, and development was performed as described above.
In vitro macrophage lysis assay. The murine macrophage cell line J774A.1 (ATCC TIB-67) was maintained in 96-well plates. Purified PA and LF (LeTx) were added simultaneously to anti-LF monoclonal or polyclonal antibody and incubated for 30 min at 4°C. The mixture was then applied to J774A cells (4 x 104 cells/well) at the following final concentrations: PA, 0.4 μg/ml; LF, 0.2 μg/ml; and anti-LF MAb, 2.5 μg/ml to 0.01 μg/ml. After incubation for 3 h at 37°C, the cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (1.5 mg/ml) for 1 h at 37°C, and the viability was determined by measuring the absorbance at 540 nm. The percentage of cells surviving toxin challenge (0.4 μg/ml PA plus 0.2 μg/ml LF) was determined as follows: (average for test wells – average for toxin-only wells) x 100%/average for no-toxin wells. The 50% inhibitory concentration (IC50) was defined as the concentration of antibody needed to protect 50% of the macrophages.
Affinity determination. Affinity constants were determined by surface plasmon resonance using a BIA coreX (Biacore). The purified LF was conjugated to a flow cell of a CM5 chip according to the manufacturer's instructions. Three concentrations of antibody were introduced into two flow cells for a defined period of time, using a flow cell conjugated with an irrelevant protein (human angiopoietin-2) as a reference signal. Curves were fitted to a 1:2 stoichiometry of binding, and the dissociation constant (Kd) was calculated from the curves using the software BIAevaluation provided by the manufacturer.
Epitope mapping. To determine the epitope recognized by the LF-neutralizing MAbs, six deletion mutants (L1, L2, L3, L4, L5, and L6) of LF were constructed from pBS1-1 LF by PCR. The L1, L2, and L3 mutants were synthesized using 1-3F as the 5' primer and 1R, 2R, or 3R as the 3' primer. The L4 mutant was synthesized using 4F as the 5' primer and 4R as the 3' primer. To construct the L5 mutant, two PCRs were performed using the 5' primer 1-3F and the 3' primer 5R or the 5' primer 5F and the 3' primer 2R, and then the resulting two PCR products were subjected to recombinant PCR using the 5' primer 1-3F and the 3' primer 2R. To construct the L6 mutant, two PCRs were performed using the 5' primer 1-4F and the 3' primer 6R or the 5' primer 6F and the 3' primer 4R, and then the resulting two PCR products were recombined by recombinant PCR using the 5' primer 1-3F and the 3' primer 4R. The final PCR products were digested with BamHI and XbaI and were subcloned into the BamHI-XbaI sites of the pBS1-2 expression vector (Aprogen, Korea). The sequences of the PCR primers were as follows: 1-3F, 5'-AATGGATCCATGGCGGGCGGTCATGGTGATG-3'; 1R, 5'-GATTCTAGAGGATAGATTTATTTCTTGTTCG-3'; 2R, 5'-ATTTCTAGATTAAATATCAAGTTTCAGC-3'; 3R, 5'-ATTTCTAGATTACACTACTTTCGCATCAATC-3'; 4R, 5'-ATTTCTAGATTATGAGTTAATAATGAACTTAA-3'; 4F, 5'-ATTGGATCCATGAAGAAAGATGACATAATT-3'; 5F, 5'-GGAAGAACTTAAAGATCAAAAGAAAGATGACATA-3'; 5R, 5'-TATGTCATCTTTCTTTTGATCTTTAAGTTCTTCC-3'; 6F, 5'-GATTCCTATTGAGCCACAACCATATGATATTAATC-3'; and 6R, 5'-GATTAATATCATATGGTTGTGGCTCAATAGGAATC-3'.
The resulting six deletion mutants of LF were expressed in E. coli HB2151 and purified by affinity chromatography on an AP1-conjugated Sepharose column as described above. After the size and integrity of each purified protein were confirmed by Western blot analysis using AP1 antibody, the same amounts of the LF proteins were subjected to slot blot analysis. Briefly, 1 μg of the native protein or a mutant protein was absorbed onto a nitrocellulose membrane. After blocking with 2% bovine serum albumin, the membrane was incubated with 5B13B1, 3C16C3, or AP1 (1 μg/ml), followed by HRP-conjugated goat anti-mouse IgG (Fc-specific) antibody, and the protein bands were detected by chemiluminescence using an ECL kit (Intron, Korea).
For fine epitope mapping, four peptides from domain III were synthesized and conjugated to keyhole limpet hemocyanin. The sequences of the peptides were as follows: R2, Ac-HSLSQEEKELLKRIQIDC; R3, Ac-SDFLSTEEKEFLKKLQIDIC; R4, Ac-DSLSEEEKELLNRIQVDSC; and R5, Ac-NPLSEKEKEFLKKLKLDIC. Binding of the MAbs to each peptide was determined by an indirect ELISA using 200 ng of the peptide-keyhole limpet hemocyanin conjugate as a coating antigen. A peptide (Ac-NKIKSALLSTNKAVVSLSNC) from the F protein of respiratory syncytial virus was used as a control.
In vivo protection assay. The in vivo toxin-neutralizing activity of the 5B13B1 antibody was evaluated using female Fisher 344 rats weighing 120 to 250 g at 6 weeks of age. Rats were anesthetized by intraperitoneal injection with Avertin (2,2,2-tribromoethyl alcohol [Aldrich]-tert-amyl alcohol; 240 mg/kg; Sigma-Aldrich). Eighty micrograms of the purified PA and 40 μg of the purified LF (LeTx) caused death in the rats within 100 min.
For the in vivo neutralization assay, LeTx (80 μg PA plus 40 μg LF) was preincubated with 42.4 μg (twice the IC50; 1.28 molar equivalents of LF) of 5B13B1 or control (anti-glutathione S-transferase [GST] MAb) antibody for 30 min at 4°C, and the mixture was injected intravenously into five rats. To evaluate the pre- and postexposure prophylactic efficacy, 5B13B1 antibody was intravenously administered at different times before and after LeTx administration. Rats were monitored for 24 h.
Measurement of the in vivo half-life of 5B13B1 in rats. The clearance of 5B13B1 antibody from the circulation of Fisher rats was determined by intravenous injection of 50 μg 5B13B1 into three rats. Blood samples were drawn at different times postinjection, and the antibody in the serum was quantitated by ELISA.
RESULTS AND DISCUSSION
Generation and selection of LeTx-neutralizing MAbs. To generate anti-LF antibodies, recombinant LF was produced from E. coli and injected into BALB/c mice as described above. After the second immunization with LF, the LeTx-neutralizing activity of the immune sera was monitored by an in vitro macrophage lysis assay using J774A.1 murine macrophage cells. A 20-fold dilution of the immune sera protected approximately 60% of the cells, while normal serum did not (data not shown). Hybridomas were screened for MAbs showing both LF-binding and LeTx-neutralizing activities. Finally, two MAbs that showed potent LeTx-neutralizing activity, 5B13B1 [IgG1()] and 3C16C3 [IgG1()], were selected for further analysis.
In vitro macrophage lysis assay of MAbs. To precisely evaluate the LeTx-neutralizing activity of MAbs 5B13B1 and 3C16C3, the antibodies were purified and preincubated with the toxin (0.4 μg/ml PA plus 0.2 μg/ml LF) for an in vitro macrophage lysis assay. As shown in Fig. 1, the two MAbs displayed neutralizing activity in a dose-dependent manner (Fig. 1). The deduced IC50s of 5B13B1 and 3C16C3 were approximately 0.21 μg/ml and 0.63 μg/ml, respectively.
The neutralizing activity of 5B13B1 was also evaluated before and after toxin challenge (0.4 μg/ml PA plus 0.2 μg/ml LF). When administered after toxin challenge, a concentration of 5B13B1 that was four times greater than its IC50 (0.83 μg/ml) conferred protection to 60% of the cells even 30 min after the challenge. When administered 60 min before toxin challenge, the same concentration of 5B13B1 (0.83 μg/ml) conferred protection to 70% of the cells (Fig. 2). This result suggests that the MAbs could be used as both prophylactics and therapeutics against anthrax poisoning.
Characterization of neutralizing MAbs. The affinities of 5B13B1 and 3C16C3 were determined using a Biacore instrument. The Kd of 5B13B1 for LF (2.62 nM) was approximately one-third that of 3C16C3 (8.18 nM).
A competition binding assay using biotinylated 5B13B1 and unlabeled 5B13B1 or 3C16C3 as a competing antibody showed that 3C16C3 efficiently competed with 5B13B1 in LF binding, although it was less efficient than 5B13B1 (Fig. 3). Considering that the affinity of 3C16C3 for LF is lower than that of 5B13B1, it is likely that these two antibodies recognize the same epitope or closely overlapping epitopes.
To determine the epitopes recognized by 5B13B1 and 3C16C3, four deletion mutants of LF (L1, L2, L3, and L4) were produced in E. coli (Fig. 4A) and tested for antibody binding by slot blot analysis (Fig. 4B). Both antibodies bound to three of the mutants (L2, L3, and L4) but not to L1. As domain III was present in the first three mutants and was not present in L1, it is likely that the epitopes recognized by the antibodies are on domain III of LF. To confirm this result, two additional mutants, L5 (domain I plus domain III) and L6 (complete LF without domain III), were constructed and subjected to slot blot analysis. As expected, both antibodies bound to L5 but not to L6 (Fig. 4B). However, the reactivity of 3C16C3 (Fig. 4B, lane 2) to the wild-type and mutant LF proteins was weaker than that of 5B13B1 (lane 1) when the same amount of proteins was used in the immunoblot analysis (lane 3), indicating that the affinity of 3C16C3 for LF is lower than that of 5B13B1. This is consistent with the Biacore data.
Domain III of LF comprises four tandem repeats, R2, R3, R4, and R5 (17). For fine epitope mapping, four peptides corresponding to each repeat were synthesized, and their reactivities with 5B13B1 and 3C16C3 were analyzed by ELISA. Only the R4 peptide bound to both of the MAbs (Fig. 4C) and completely inhibited the binding of the MAbs to LF (Fig. 4D). These data clearly demonstrate that 5B13B1 and 3C16C3 recognize the same or closely overlapping epitopes on the R4 repeat sequence of domain III. Based on the findings obtained, we concluded that the difference in the neutralizing activities of the antibodies was due to the difference in their affinities. Therefore, 5B13B1, which had the highest affinity, was chosen for further in vivo study.
In vivo protection assay. The in vivo LeTx-neutralizing activity of 5B13B1 was evaluated in Fisher 344 rats. Six rats were challenged with mixture of LeTx (80 μg PA plus 40 μg LF) and control antibody (anti-GST mouse MAb) or a mixture of LeTx and 5B13B1 at twice its IC50 (42 μg; 1.28 molar equivalents of LF). The results showed that 5B13B1 protected 100% of the animals from death caused by challenge with LeTx, while all six control rats were dead within 80 min, with an average time till death of 58 ± 9 min (Fig. 5). To investigate the efficacy of postexposure prophylaxis, 5B13B1 at twice its IC50 was intravenously administered to three groups of four Fisher rats at 5, 15, or 30 min after the toxin challenge. 5B13B1 protected 100% of the rats from death when it was administered 5 min after exposure to LeTx. When 5B13B1 was administered 15 min after exposure, 50% of the rats were protected, whereas all four rats died when the antibody was administered 30 min after toxin challenge (Fig. 6). The two rats that received antibody 15 min after toxin challenge and died had a delay in the time till death (average, 337 min) compared with the control group that received control antibody (anti-GST MAb) (average, 56 min).
The preexposure prophylactic efficacy of 5B13B1 was also evaluated. Four Fisher 344 rats were given a single intravenous injection of 5B13B1 (four times the IC50; 84.8 μg) at 0, 1, 3, or 5 days prior to LeTx challenge. The dose of antibody corresponded to 2.56 molar equivalents of LF. Even when the antibody was administered 5 days prior to toxin challenge, no death was observed within 24 h (Fig. 7). Considering that the in vivo half-life of 5B13B1 in Fisher rats was approximately 65 h, a dose of 5B13B1 that was approximately equal to the IC50 (21.2 μg), corresponding to 0.64 molar equivalent of the administered LF, might be sufficient to protect rats from LeTx challenge.
Regarding the structure-function relationship of LF, domain I of LF binds to PA, and domains II, III, and IV together create a long catalytic groove into which the amino terminus of MAPKK fits (11, 20). Acidic residues in domain III make specific contact with the basic residues at the amino terminus of MAPKK (11). It has been presumed that LF-neutralizing antibody binds to domain I and thus directly inhibits the binding of LF to PA to neutralize LeTx (21, 24). In this study, however, two MAbs that recognize domain III, 5B13B1 and 3C16C3, showed the highest affinity and the most potent toxin-neutralizing activity. This suggests that domain III may carry immunodominant neutralization epitopes. The possible mechanisms of neutralization by these MAbs include (i) disruption of the binding of LF to PA owing to a structural change in LF induced by antibody binding, (ii) inhibition of toxin endocytosis, (iii) inhibition of the translocation of LF to the cytosol through a PA channel, and/or (iv) inhibition of the catalytic action of LF. The exact neutralization mechanism of 5B13B1 remains to be elucidated.
In summary, this is the first report that an MAb (5B13B1) binding to domain III of LF has potent neutralizing activity against LeTx in vitro and in vivo. The antibody showed pre- and postexposure prophylactic effects in an in vivo protection assay using rats. Thus, this LF-neutralizing antibody may be useful in the prophylaxis of anthrax poisoning.
ACKNOWLEDGMENTS
This work was supported by a grant (grant M1020804001-02B2404-00310; "Development of Countermeasures against Chemical and Biological Terrorism") from the Ministry of Commerce, Industry and Energy, Korea, and in part by a grant (grant AO50260; "Center for therapeutic antibody") from the Ministry of Health and Welfare.
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Laboratory of Antibody Engineering, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejon 305-333, South Korea
ABSTRACT
Lethal factor (LF) is a component of anthrax lethal toxin (LeTx). We generated anti-LF murine monoclonal antibodies (MAbs) that show LeTx-neutralizing activity in vitro and in vivo. Anti-LF MAbs were generated by immunization with recombinant LF, and the MAbs showing LeTx-neutralizing activity in vitro were selected. Two MAbs with the highest affinities, 5B13B1 (dissociation constant [Kd], 2.62 nM) and 3C16C3 (Kd, 8.18 nM), were shown to recognize the same or closely overlapping epitopes on domain III of LF. The 50% inhibitory concentration of 5B13B1 (0.21 μg/ml) was approximately one-third that of 3C16C3 (0.63 μg/ml) in the in vitro LeTx-neutralization assay. The 5B13B1 antibody, which had the highest neutralizing activity, provided perfect protection against LeTx challenge in an in vivo LeTx neutralization assay using Fisher 344 rats. In addition, the antibody showed pre- and postexposure prophylactic effects in the animal experiments. This is the first report that an MAb binding to domain III of LF has neutralizing activity against LeTx. The 5B13B1 antibody may be useful in prophylaxis against anthrax poisoning.
INTRODUCTION
Bacillus anthracis, the causative agent of anthrax, is a spore-forming, gram-positive bacterium. Infection by inhalation of the spores of B. anthracis can result in a mortality rate of up to 80% if the infection is left untreated. This organism was one of the first biological warfare agents to be developed and continues to be a major threat in this regard (8, 14, 25). Anthrax is delivered as inert spores, which germinate into replicating bacteria that produce anthrax toxins. During a systemic infection, B. anthracis can replicate to produce high levels in the bloodstream. At this point in the infection, although aggressive antibiotic therapy can prevent bacterial growth, infected individuals can still die, most probably as a result of high concentrations of bacterial toxins already accumulated in the body (21). Thus, an ideal counterattack should include both killing the B. anthracis with antibiotics and neutralizing the toxin (5). In fact, a combination of antibiotic and immunoglobulin therapy was shown to be more effective than antibiotic treatment alone in a rodent anthrax model (9).
Anthrax toxin consists of three components, designated protective antigen (PA), lethal factor (LF), and edema factor (EF), that together form a tripartite protein exotoxin (15). LF along with PA forms a toxin referred to as lethal toxin (LeTx). After binding to the cell surface receptors TEM8 or CMG2, PA is cleaved into two fragments by a furin-like protease. This allows the carboxy-terminal fragment, PA63, to heptamerize and bind LF or EF. The resulting complexes of (PA63)7 with LF or EF are taken up into cells by receptor-mediated endocytosis and move to an endosomal compartment. The translocation of bound LF or EF into the cytosol is promoted by the structural change of PA induced by an acidic environment (3, 23).
LF is the major virulence factor and is responsible for shock and death (7, 18-20). LF is a zinc-dependent protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near their amino termini. This leads to the inhibition of one or more signaling pathways and thus causes lysis of macrophages (17, 24). LF also offers B. anthracis an efficient mechanism to evade the innate immune response by inhibiting interferon regulatory factor 3 activation by lipopolysaccharide and subsequent cytokine production through bacterial membrane components. In addition, LF severely impairs the function of dendritic cells by disrupting the mitogen-activated kinase intracellular signaling network (1, 6).
Some studies have shown that passive transfer of a neutralizing polyclonal antibody or monoclonal antibody (MAb) can protect cells against anthrax toxin or bacterial challenge (2, 4, 10, 12, 13, 22, 24). The protective efficacy of neutralizing antibody was greatly enhanced by a combination of a PA-neutralizing antibody and an LF-neutralizing antibody (4). Nevertheless, PA has been the primary target for passive protection, as the current approved anthrax vaccine consists principally of PA. In contrast, only a couple of LF-neutralizing MAbs have been described (13, 24). These MAbs were shown to interfere with the binding of LF to PA, but neither their epitope specificities nor the neutralization mechanism was studied.
In this study, we generated LF-neutralizing MAbs that specifically bind domain III of LF, and we confirmed their protective efficacy by performing in vitro and in vivo LeTx neutralization assays. One of the MAbs (5B13B1) protected Fisher 344 rats from LeTx challenge when it was administered before or after exposure to LeTx. The mechanism of neutralization by this MAb is discussed below.
MATERIALS AND METHODS
Expression and purification of PA and LF. The DNA encoding PA was prepared by digestion of pT7-PA (kindly provided by W. K. Seong at the Korea Center for Disease Control and Prevention) with BamHI and SalI and was ligated into the BamHI-SalI sites of pBS1-1 (Aprogen, Korea), which contains an S1 tag at the 5' end of the fused gene (16), to construct pBS1-1-PA. The DNA encoding LF was synthesized by PCR from pXO1 DNA (kindly provided by W. K. Seong. Sung at the Korea Center for Disease Control and Prevention) using a 5' primer (5'-CGTGGATCCATGGCGGGCGGTCATGGTGATG-3') and a 3' primer (5'-GATTCTAGATTATGAGTTAATAATGAAC-3'). The PCR products were digested with BamHI and XbaI and ligated into the BamHI-XbaI sites of pBS1-1 to construct pBS1-1-LF.
To express PA and LF in bacteria, each of the expression plasmids was introduced into Escherichia coli HB2151, and the fresh transformants were grown in 2x YT medium supplemented with ampicillin (100 μg/ml) at 37°C. Induction of gene expression by addition of 1 mM isopropyl--D-thiogalactopyranoside (IPTG) was performed at 30°C for 4 h. The proteins were purified from cleared bacterial lysates by affinity chromatography on an AP1 (murine anti-S1-tagged MAb)-conjugated Sepharose column (Aprogen, Korea). To remove the S1 tag from the S1-tagged LF (S1-LF) and PA (S1-PA), purified S1-LF and S1-PA were incubated with thrombin (1 U/100 μg of fusion protein) at room temperature for 2 h. The fractions containing the toxin protein were separated by fast protein liquid chromatography on a Superose 6 column (Amersham). The purity of the purified proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Generation of LF-neutralizing MAbs. BALB/c mice were immunized with the purified LF by subcutaneous injections. For the first injection, 20 μg of the immunogen was emulsified with complete Freund's adjuvant (Sigma). Three subcutaneous booster injections of LF in incomplete Freund's adjuvant (Sigma) were given every 2 weeks. Fourteen days later, the mice received the last intravenous injection without adjuvant. Splenocytes were collected 3 days later and were immortalized by fusion with mouse myeloma cells (F0; ATCC CRL-1645). The cells were grown in 96-well plates in HAT medium, and the culture medium was screened by an enzyme-linked immunosorbent assay (ELISA) using the purified LF as a coating antigen. The positive clones were screened for LeTx-neutralizing activity by an in vitro macrophage lysis assay. Hybridomas showing LF-binding activity and LeTx-neutralizing activity were subcloned by limiting dilution. MAb was purified from the culture supernatant of hybridomas grown in serum-free medium by affinity chromatography on a protein G-Sepharose column (Amersham).
ELISA. To detect the antibody secreted by hybridomas, each well was coated with the purified LF (2 μg/ml). Then 100 μl of the culture supernatant was added to each well. After three washes, 50 μl of horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (IgG) (Fc-specific) antibody (Pierce) was added to each well and incubated for 1 h at 37°C. After washing, 0.2 M citrate-PO4 buffer (pH 5.0) containing 0.04% ortho-phenylenediamine dihydrochloride and 0.012% H2O2 was added to each well. The reaction was stopped by adding 2.5 M H2SO4, and the absorbance was determined at 492 nm using an ELISA reader (SOFTmaxPRO; Molecular Devices, United States).
For the competition binding assay, each well was coated with the purified LF (100 ng) and blocked with 2% bovine serum albumin for 1 h at 37°C. After washing, biotinylated 5B13B1 and serially diluted unlabeled 5B13B1 or 3C16C3 were added to each well, and the plate was incubated at 37°C for 1 h. After washing, streptavidin-HRP (Pierce) was added for 1 h, and development was performed as described above.
In vitro macrophage lysis assay. The murine macrophage cell line J774A.1 (ATCC TIB-67) was maintained in 96-well plates. Purified PA and LF (LeTx) were added simultaneously to anti-LF monoclonal or polyclonal antibody and incubated for 30 min at 4°C. The mixture was then applied to J774A cells (4 x 104 cells/well) at the following final concentrations: PA, 0.4 μg/ml; LF, 0.2 μg/ml; and anti-LF MAb, 2.5 μg/ml to 0.01 μg/ml. After incubation for 3 h at 37°C, the cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (1.5 mg/ml) for 1 h at 37°C, and the viability was determined by measuring the absorbance at 540 nm. The percentage of cells surviving toxin challenge (0.4 μg/ml PA plus 0.2 μg/ml LF) was determined as follows: (average for test wells – average for toxin-only wells) x 100%/average for no-toxin wells. The 50% inhibitory concentration (IC50) was defined as the concentration of antibody needed to protect 50% of the macrophages.
Affinity determination. Affinity constants were determined by surface plasmon resonance using a BIA coreX (Biacore). The purified LF was conjugated to a flow cell of a CM5 chip according to the manufacturer's instructions. Three concentrations of antibody were introduced into two flow cells for a defined period of time, using a flow cell conjugated with an irrelevant protein (human angiopoietin-2) as a reference signal. Curves were fitted to a 1:2 stoichiometry of binding, and the dissociation constant (Kd) was calculated from the curves using the software BIAevaluation provided by the manufacturer.
Epitope mapping. To determine the epitope recognized by the LF-neutralizing MAbs, six deletion mutants (L1, L2, L3, L4, L5, and L6) of LF were constructed from pBS1-1 LF by PCR. The L1, L2, and L3 mutants were synthesized using 1-3F as the 5' primer and 1R, 2R, or 3R as the 3' primer. The L4 mutant was synthesized using 4F as the 5' primer and 4R as the 3' primer. To construct the L5 mutant, two PCRs were performed using the 5' primer 1-3F and the 3' primer 5R or the 5' primer 5F and the 3' primer 2R, and then the resulting two PCR products were subjected to recombinant PCR using the 5' primer 1-3F and the 3' primer 2R. To construct the L6 mutant, two PCRs were performed using the 5' primer 1-4F and the 3' primer 6R or the 5' primer 6F and the 3' primer 4R, and then the resulting two PCR products were recombined by recombinant PCR using the 5' primer 1-3F and the 3' primer 4R. The final PCR products were digested with BamHI and XbaI and were subcloned into the BamHI-XbaI sites of the pBS1-2 expression vector (Aprogen, Korea). The sequences of the PCR primers were as follows: 1-3F, 5'-AATGGATCCATGGCGGGCGGTCATGGTGATG-3'; 1R, 5'-GATTCTAGAGGATAGATTTATTTCTTGTTCG-3'; 2R, 5'-ATTTCTAGATTAAATATCAAGTTTCAGC-3'; 3R, 5'-ATTTCTAGATTACACTACTTTCGCATCAATC-3'; 4R, 5'-ATTTCTAGATTATGAGTTAATAATGAACTTAA-3'; 4F, 5'-ATTGGATCCATGAAGAAAGATGACATAATT-3'; 5F, 5'-GGAAGAACTTAAAGATCAAAAGAAAGATGACATA-3'; 5R, 5'-TATGTCATCTTTCTTTTGATCTTTAAGTTCTTCC-3'; 6F, 5'-GATTCCTATTGAGCCACAACCATATGATATTAATC-3'; and 6R, 5'-GATTAATATCATATGGTTGTGGCTCAATAGGAATC-3'.
The resulting six deletion mutants of LF were expressed in E. coli HB2151 and purified by affinity chromatography on an AP1-conjugated Sepharose column as described above. After the size and integrity of each purified protein were confirmed by Western blot analysis using AP1 antibody, the same amounts of the LF proteins were subjected to slot blot analysis. Briefly, 1 μg of the native protein or a mutant protein was absorbed onto a nitrocellulose membrane. After blocking with 2% bovine serum albumin, the membrane was incubated with 5B13B1, 3C16C3, or AP1 (1 μg/ml), followed by HRP-conjugated goat anti-mouse IgG (Fc-specific) antibody, and the protein bands were detected by chemiluminescence using an ECL kit (Intron, Korea).
For fine epitope mapping, four peptides from domain III were synthesized and conjugated to keyhole limpet hemocyanin. The sequences of the peptides were as follows: R2, Ac-HSLSQEEKELLKRIQIDC; R3, Ac-SDFLSTEEKEFLKKLQIDIC; R4, Ac-DSLSEEEKELLNRIQVDSC; and R5, Ac-NPLSEKEKEFLKKLKLDIC. Binding of the MAbs to each peptide was determined by an indirect ELISA using 200 ng of the peptide-keyhole limpet hemocyanin conjugate as a coating antigen. A peptide (Ac-NKIKSALLSTNKAVVSLSNC) from the F protein of respiratory syncytial virus was used as a control.
In vivo protection assay. The in vivo toxin-neutralizing activity of the 5B13B1 antibody was evaluated using female Fisher 344 rats weighing 120 to 250 g at 6 weeks of age. Rats were anesthetized by intraperitoneal injection with Avertin (2,2,2-tribromoethyl alcohol [Aldrich]-tert-amyl alcohol; 240 mg/kg; Sigma-Aldrich). Eighty micrograms of the purified PA and 40 μg of the purified LF (LeTx) caused death in the rats within 100 min.
For the in vivo neutralization assay, LeTx (80 μg PA plus 40 μg LF) was preincubated with 42.4 μg (twice the IC50; 1.28 molar equivalents of LF) of 5B13B1 or control (anti-glutathione S-transferase [GST] MAb) antibody for 30 min at 4°C, and the mixture was injected intravenously into five rats. To evaluate the pre- and postexposure prophylactic efficacy, 5B13B1 antibody was intravenously administered at different times before and after LeTx administration. Rats were monitored for 24 h.
Measurement of the in vivo half-life of 5B13B1 in rats. The clearance of 5B13B1 antibody from the circulation of Fisher rats was determined by intravenous injection of 50 μg 5B13B1 into three rats. Blood samples were drawn at different times postinjection, and the antibody in the serum was quantitated by ELISA.
RESULTS AND DISCUSSION
Generation and selection of LeTx-neutralizing MAbs. To generate anti-LF antibodies, recombinant LF was produced from E. coli and injected into BALB/c mice as described above. After the second immunization with LF, the LeTx-neutralizing activity of the immune sera was monitored by an in vitro macrophage lysis assay using J774A.1 murine macrophage cells. A 20-fold dilution of the immune sera protected approximately 60% of the cells, while normal serum did not (data not shown). Hybridomas were screened for MAbs showing both LF-binding and LeTx-neutralizing activities. Finally, two MAbs that showed potent LeTx-neutralizing activity, 5B13B1 [IgG1()] and 3C16C3 [IgG1()], were selected for further analysis.
In vitro macrophage lysis assay of MAbs. To precisely evaluate the LeTx-neutralizing activity of MAbs 5B13B1 and 3C16C3, the antibodies were purified and preincubated with the toxin (0.4 μg/ml PA plus 0.2 μg/ml LF) for an in vitro macrophage lysis assay. As shown in Fig. 1, the two MAbs displayed neutralizing activity in a dose-dependent manner (Fig. 1). The deduced IC50s of 5B13B1 and 3C16C3 were approximately 0.21 μg/ml and 0.63 μg/ml, respectively.
The neutralizing activity of 5B13B1 was also evaluated before and after toxin challenge (0.4 μg/ml PA plus 0.2 μg/ml LF). When administered after toxin challenge, a concentration of 5B13B1 that was four times greater than its IC50 (0.83 μg/ml) conferred protection to 60% of the cells even 30 min after the challenge. When administered 60 min before toxin challenge, the same concentration of 5B13B1 (0.83 μg/ml) conferred protection to 70% of the cells (Fig. 2). This result suggests that the MAbs could be used as both prophylactics and therapeutics against anthrax poisoning.
Characterization of neutralizing MAbs. The affinities of 5B13B1 and 3C16C3 were determined using a Biacore instrument. The Kd of 5B13B1 for LF (2.62 nM) was approximately one-third that of 3C16C3 (8.18 nM).
A competition binding assay using biotinylated 5B13B1 and unlabeled 5B13B1 or 3C16C3 as a competing antibody showed that 3C16C3 efficiently competed with 5B13B1 in LF binding, although it was less efficient than 5B13B1 (Fig. 3). Considering that the affinity of 3C16C3 for LF is lower than that of 5B13B1, it is likely that these two antibodies recognize the same epitope or closely overlapping epitopes.
To determine the epitopes recognized by 5B13B1 and 3C16C3, four deletion mutants of LF (L1, L2, L3, and L4) were produced in E. coli (Fig. 4A) and tested for antibody binding by slot blot analysis (Fig. 4B). Both antibodies bound to three of the mutants (L2, L3, and L4) but not to L1. As domain III was present in the first three mutants and was not present in L1, it is likely that the epitopes recognized by the antibodies are on domain III of LF. To confirm this result, two additional mutants, L5 (domain I plus domain III) and L6 (complete LF without domain III), were constructed and subjected to slot blot analysis. As expected, both antibodies bound to L5 but not to L6 (Fig. 4B). However, the reactivity of 3C16C3 (Fig. 4B, lane 2) to the wild-type and mutant LF proteins was weaker than that of 5B13B1 (lane 1) when the same amount of proteins was used in the immunoblot analysis (lane 3), indicating that the affinity of 3C16C3 for LF is lower than that of 5B13B1. This is consistent with the Biacore data.
Domain III of LF comprises four tandem repeats, R2, R3, R4, and R5 (17). For fine epitope mapping, four peptides corresponding to each repeat were synthesized, and their reactivities with 5B13B1 and 3C16C3 were analyzed by ELISA. Only the R4 peptide bound to both of the MAbs (Fig. 4C) and completely inhibited the binding of the MAbs to LF (Fig. 4D). These data clearly demonstrate that 5B13B1 and 3C16C3 recognize the same or closely overlapping epitopes on the R4 repeat sequence of domain III. Based on the findings obtained, we concluded that the difference in the neutralizing activities of the antibodies was due to the difference in their affinities. Therefore, 5B13B1, which had the highest affinity, was chosen for further in vivo study.
In vivo protection assay. The in vivo LeTx-neutralizing activity of 5B13B1 was evaluated in Fisher 344 rats. Six rats were challenged with mixture of LeTx (80 μg PA plus 40 μg LF) and control antibody (anti-GST mouse MAb) or a mixture of LeTx and 5B13B1 at twice its IC50 (42 μg; 1.28 molar equivalents of LF). The results showed that 5B13B1 protected 100% of the animals from death caused by challenge with LeTx, while all six control rats were dead within 80 min, with an average time till death of 58 ± 9 min (Fig. 5). To investigate the efficacy of postexposure prophylaxis, 5B13B1 at twice its IC50 was intravenously administered to three groups of four Fisher rats at 5, 15, or 30 min after the toxin challenge. 5B13B1 protected 100% of the rats from death when it was administered 5 min after exposure to LeTx. When 5B13B1 was administered 15 min after exposure, 50% of the rats were protected, whereas all four rats died when the antibody was administered 30 min after toxin challenge (Fig. 6). The two rats that received antibody 15 min after toxin challenge and died had a delay in the time till death (average, 337 min) compared with the control group that received control antibody (anti-GST MAb) (average, 56 min).
The preexposure prophylactic efficacy of 5B13B1 was also evaluated. Four Fisher 344 rats were given a single intravenous injection of 5B13B1 (four times the IC50; 84.8 μg) at 0, 1, 3, or 5 days prior to LeTx challenge. The dose of antibody corresponded to 2.56 molar equivalents of LF. Even when the antibody was administered 5 days prior to toxin challenge, no death was observed within 24 h (Fig. 7). Considering that the in vivo half-life of 5B13B1 in Fisher rats was approximately 65 h, a dose of 5B13B1 that was approximately equal to the IC50 (21.2 μg), corresponding to 0.64 molar equivalent of the administered LF, might be sufficient to protect rats from LeTx challenge.
Regarding the structure-function relationship of LF, domain I of LF binds to PA, and domains II, III, and IV together create a long catalytic groove into which the amino terminus of MAPKK fits (11, 20). Acidic residues in domain III make specific contact with the basic residues at the amino terminus of MAPKK (11). It has been presumed that LF-neutralizing antibody binds to domain I and thus directly inhibits the binding of LF to PA to neutralize LeTx (21, 24). In this study, however, two MAbs that recognize domain III, 5B13B1 and 3C16C3, showed the highest affinity and the most potent toxin-neutralizing activity. This suggests that domain III may carry immunodominant neutralization epitopes. The possible mechanisms of neutralization by these MAbs include (i) disruption of the binding of LF to PA owing to a structural change in LF induced by antibody binding, (ii) inhibition of toxin endocytosis, (iii) inhibition of the translocation of LF to the cytosol through a PA channel, and/or (iv) inhibition of the catalytic action of LF. The exact neutralization mechanism of 5B13B1 remains to be elucidated.
In summary, this is the first report that an MAb (5B13B1) binding to domain III of LF has potent neutralizing activity against LeTx in vitro and in vivo. The antibody showed pre- and postexposure prophylactic effects in an in vivo protection assay using rats. Thus, this LF-neutralizing antibody may be useful in the prophylaxis of anthrax poisoning.
ACKNOWLEDGMENTS
This work was supported by a grant (grant M1020804001-02B2404-00310; "Development of Countermeasures against Chemical and Biological Terrorism") from the Ministry of Commerce, Industry and Energy, Korea, and in part by a grant (grant AO50260; "Center for therapeutic antibody") from the Ministry of Health and Welfare.
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