The 2β2–2β3 loop of anthrax protective antigen contains a dominant neutralizing epitope

BBRC Biochemical and Biophysical Research Communications 341 (2006) 1164–1171 www.elsevier.com/locate/ybbrc

The 2b2–2b3 loop of anthrax protective antigen contains a dominant neutralizing epitope Jun Zhang, Junjie Xu *, Guanlin Li, Dayong Dong, Xiaohong Song, Qiang Guo, Jian Zhao, Ling Fu, Wei Chen * Department of Applied Molecular Biology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dongdajie, Fengtai, Beijing 100071, China Received 15 January 2006 Available online 26 January 2006

Abstract Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, 312SFFD315, in the 2b2–2b3 loop of PA, using phagedisplayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2b2–2b3 loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax.
2006 Elsevier Inc. All rights reserved. Keywords: Anthrax toxin; Protective antigen; Epitope; Neutralizing epitope

Bacillus anthracis is the etiologic agent of anthrax in animals and humans. Two plasmid-encoded virulence factors, a poly-D-glutamic acid capsule, and an exotoxin, have been described for the bacterium. The exotoxin is composed of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). PA induces protective immunity to the infection of the bacteria and is the major component in the current anthrax vaccine (anthrax vaccine adsorbed, AVA) [1]. EF is an adenylate cyclase that disrupts water homeostasis and impairs immune function in the host [2]. LF is a Zn2+-dependent metalloproteinase that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family of protein kinase and disrupts signal transduction pathways [3,4]. PA binds to toxin receptors on the surface of the host cell and then is cleaved by *

Corresponding authors. Fax: +86 10 63815273 (J. Xu). E-mail addresses: [email protected] (J. Xu), chenwei0226@yahoo. com.cn (W. Chen). 0006-291X/$ - see front matter
2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.01.080

a Furin-like protease releasing a 20-kDa fragment (PA20) and leaving a 63-kDa portion (PA63). PA63 is capable of forming a heptamer, which has an exposed surface that binds EF and LF. Heptamer complexes enter the endocytic pathway by receptor mediated endocytosis, and upon acidification of the vesicle, the PA63 heptamer undergoes a conformation change to form a pore through which EF and LF translocation into the cytoplasm. Once in the cytoplasm, EF and LF exert toxin effect [5,6]. Two cellular receptors that bind PA have been identified as anthrax toxin receptors, one of which is a protein-encoded by the tumor endothelial marker-8 (TEM8) gene [7], the other is the human capillary morphogenesis protein 2 (CMG2) [8]. The detailed mechanism of the interaction of PA and its receptors is still on the investigation [9]. Besides being the key factor in the pathogenesis of anthrax toxin, PA also plays an important role in immunity and prophylaxis against anthrax [10]. Much of the work in the past decade on generating a next generation anthrax

J. Zhang et al. / Biochemical and Biophysical Research Communications 341 (2006) 1164–1171

vaccine with recombinant PA as the immunogen was based on the observation that antibodies to PA are crucial in the protection against exposure to virulent anthrax spores. Many investigations indicated that passive administration of polyclonal antibodies or monoclonal antibodies (MAbs) to PA can protect animals against anthrax. [11–15]. In addition to their well-defined toxin-neutralizing activity, anti-PA antibodies have also been shown to exhibit antispore activities [16,17]. On the other hand, in some studies, the MAbs against PA increased LT-mediated killing of murine macrophages [18,19]. This suggests that some epitopes on PA may be responsible for generation of toxin-enhancing antibodies, possibly decreasing the neutralizing capacity of toxin-neutralizing antibodies. Therefore, to develop the next-generation anthrax vaccine, further investigation of the immunological mechanism of PA is necessary, and definition of domiant antigenic epitopes in PA is an important step. In this report, a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) was generated to analyze the neutralizing epitopes of PA, and a dominant neutralizing epitope was fine mapped to the chymotrypsin-sensitive site, 312SFFD315, in the 2b2–2b3 loop of PA. Materials and methods Proteins. Recombinant PA and LF were expressed in Escherichia coli as described previously [20,21]. Fragments of PA comprising one or more domains were also expressed in E. coli, by changing PA gene in expression plasmid pAS-PA[20] with sequences encoding different PA domains. These fragments include PAD4 (domain 4, amino acid 596–735, and 17 kDa), PAD3–4(domain 3–4, amino acid 489–735, and 28 kDa), PAD1–3(domain 1–3, amino acid 1–607, and 68 kDa), PAD1 (domain 1, amino acid 1–258, 29 kDa).The recombinant proteins were purified to at least 90% pure by chromatography. Production and identification of toxin-neutralizing MAbs. Anti-PA MAbs were prepared according to the standard hybridoma technique. Spleen cells from BALB/c mouse immunized with recombinant PA was fused with mouse myeloma cells (SP2/0). Hybridoma clones were screened by enzyme-linked immunosorbent assay (ELISA) with PA as coated antigen. Positive hybridomas were then screened their ability to neutralize lethal toxin activities in vitro on a toxin-sensitive cell line as described below. Clones exhibiting toxin neutralization were developed further to get stable cell lines. Ascites from each hybridoma were produced in mice and monoclonal antibodies were purified by Protein G chromatography (Amersham). Toxin-neutralizing monoclonal antibodies obtained were then characterized for antibody subclass using ImmunoType Kit (Sigma) and domains of PA they recognized by ELISA and Western blot analysis. In vitro toxin neutralization assay. J774A.1 cells were plated in 96-well flat bottom tissue culture plates at a density of 1 · 105 cells/well in 100 ll DMEM supplemented with 10% fetal calf serum and incubated overnight. The next day, PA (100 ng/ml) and LF (100 ng/ml) were pre-incubated with a range of dilutions of each antibody sample for 60 min at 37 C in a working volume of 100 ll of the same medium. This 100 ll volume was subsequently transferred into each cell wells to replace the original medium. The culture plates were incubated for 3 h at 37 C. Cell viability was determined by MTT dye in which MTT dissolved in the medium was added to each well at a final concentration of 0.5 mg/ml. Cells were incubated for another 30 min at 37 C. The medium was replaced by 0.5% (w/v) SDS, 25 mM HCl in 90% isopropyl alcohol, the plate was vortexed, and the absorbance readings at 570 nm were measured with a microplate reader. All experiments were done in triplicate. The purified rabbit antiPA polyclonal antibodies (RAb) were used as control. Cell viability was

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analyzed by using the average A570 of pairs of wells receiving the toxin (PA + LF) plus the antibody or the toxin alone. The antibody dilution that resulted in an optical density signal of 0.1 was used as a measure of the toxin neutralization assay (TNA) titer. Competitive binding assays to identify the epitopes recognized by MAbs. Purified MAbs were conjugated with peroxidase using EZ-LINK plus activated peroxidase kit (Pierce), according to the manufacturer’s directions. Competition between MAbs was measured by ELISA, using each purified MAb to compete for the binding of a limiting concentration of HRP-MAb. In brief, 96-well plates were coated with PA, 50 ll of unconjugated MAb (MAb1) at a concentration of 10 lg/ml and 50 ll of HRP-MAb (MAb2) were added to each well, and the plate was incubated for 1 h at 37 C. Control wells contained 50 ll of HRP-MAb and 50 ll of diluting buffer. The plates were washed and 100 ll of developing solution (TMB/H2O2) was added to each well. After approximately 10 min, 2 M H2SO4 was added and the plates were read at 450 nm. Dilutions for MAb2 were selected to give an adsorption value about 1.0 for control wells. The observed A450 values were used to calculate the percentage that MAb1 blocked the binding of the MAb2 by the following formula: % blocking = (1
test well A450/control well A450) · 100. MAbs showed >50% of blocking were considered recognize a same potential epitope. The same assay was used to identify the competition between rabbit anti-PA polyclonal antibodies and MAbs, with the twofold serially diluted polyclonal antibodies beginning from 10 lg/ml to block the binding of HRP-MAbs to PA. Epitope mapping by phage display technique. The PhD-12 and PhDC7C Phage Display Peptide Library Kit (New England Biolabs) were used for the epitope mapping. Briefly, the MAb at 100 lg/ml in carbonate buffer (0.1 M NaHCO3, pH 8.6) was immobilized in 96-well plates for 16 h at 4 C. The coating solution was removed, and each well was filled with blocking buffer (0.1 M NaHCO3, pH 8.6, 5 mg/ml BSA, and 0.02% NaN3) for 1 h at 4 C. Then the blocking solution was discarded and each plate was washed rapidly six times with TBST (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.5% [v/v] Tween 20). Ten microliters of original phage library was diluted with 100 ll TBST and pipetted into coated plate and rocked gently for 1 h at room temperature. Then unbound phage was discarded by pouring off and plates were washed 10 times with TBST. Bound phage was eluted with 100 ll of 0.2 M glycine–HCl (pH 2.2), 1 mg/ ml BSA for 10 min with subsequent neutralization with 15 ll of 1 M Tris– HCl (pH 9.1). A small amount of the eluted phage (about 1 ll) was tittered by standard microbiological techniques. The remaining eluate was added to exponentially growing E. coli ER2738 cells and amplified by incubation at 37 C for 5 h. To precipitate the phage, a one-sixth volume of PEG8000/NaCl (20% [w/v] polyethylene glycol-8000, 2.5 M NaCl) was added to the culture. Phages were finally resuspended in 100 ll TBS, and were titered. This biopanning procedure was repeated twice, using 1011 of the amplified phage as an input for each biopanning process. The stringency of selection during panning was gradually increased with each round by raising the Tween 20 concentration stepwise from 0.5%, 0.8%, and finally to a maximum of 1%. Competition experiments of phage clones. Phage clones selected from the third round of panning were amplified and purified for sequent experiments. ELISA plates were coated with 2 lg/ml MAb in carbonate buffer (as above) at 4 C overnight. Then the coated solution was shaken out and each well was filled completely with blocking buffer. The plates were incubated 1 h at 4 C, then the blocking buffer was removed and each plate was washed four times with TBST. The percentage of Tween 20 should be the same as the concentration used in the panning wash steps. We carried out twofold serial dilutions of the phage clones beginning with equimultiple volume of amplified phages supernatant and the blocking buffer in 150 ll per well in which PA were added to a concentration of 5 lg/ml. Simultaneously, we left the last well of every row as control (without phages). After incubated at 37 C for 1 h, the plates were washed four times with TBST and incubated with 100 ll of rabbit anti-PA polyclonal antibodies at 1 lg/ml for 1 h at 37 C. The plates were washed and then 100 ll HRP conjugated goat anti-rabbit IgG (Sigma) at 1:20,000 was added to each well. After incubated at 37 C for 1 h, the plates were washed again and 100 ll of developing solution (TMB/H2O2) was added.

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After approximately 10 min, 2 M H2SO4 was added and the plates were analyzed using a microplate reader at a wavelength of 450 nm. The results were calculated as mean absorbance (A450) of duplicate wells for one sample. The percentage that PA blocked the binding of the MAb and the phage clones by the following formula: % blocking = (1
test well A450/ control well A450) · 100. Characterization of phage inserts. The positive phage clones obtained from competition experiment were amplified and purified as above but in the last step the phage pellets were thoroughly suspended in 100 ll iodide buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, and 4 M NaI) and then 250 ll ethanol was added. After incubated 10 min at room temperature, the phages were centrifuged for 10 min. Then the supernatant was discarded and the pellet was washed in 70% ethanol and dried briefly under vacuum. Finally the pellet was suspended in 30 ll TE buffer (10 mM Tris– HCl, pH 8.0, 1 mM EDTA). The phage inserts were sequenced and the results were aligned to find a consensus. Phage immunization. One of phage clones containing the consensus sequence was amplified and used directly as immunogen in BALB/c mice. Another two groups of mice were immunized with recombinant PA and control phages (amplified PhD-12 Library), respectively. See Table 2 for immunization schedule and procedure. Seven days after the last injection, serum samples were drawn from mice, and anti-PA antibody was detected by ELISA with PA as coated antigen. Expression of the PA mutant. PA mutant (312SFFD315 deleted) plasmid was constructed from parental plasmid pAS-PA [20] through PCR. Two primers were used to amplify the region covering the unique PstI and HindIII sites in the PA coding region on pAS-PA. Primer1 is (5 0 -aggagaaccggttattaaatgaatc-3 0 ), and Prime2 is (5 0 -taaatcctgcagata cactcccaccaatcgcatgcacttctgcattt-3 0 ) which contains the desired mutation. The PCR product and pAS-PA were digested with PstI and HindIII, respectively, prior to ligation. The construct was verified by DNA sequencing and transformed to E. coli DH5a host strain. PA mutant (PAm) was purified from the periplasm of cells through a purification procedure including anion-exchange chromatography, hydrophobic chromatography. Purified PAm was analyzed by SDS–PAGE and Western blot analysis. Cytotoxicity assays of the PA mutant. J774A.1 cells were plated at a density of about 1 · 105 cells/well and incubated overnight. The next day growth medium was removed by cautious aspiration and replaced with 100 ll of new medium containing 100 ng/ml LF and a dilution series of PA or PAm from 10 lg/ml to 0, where each tested dilution was made in triplicate wells. Cells were incubated at 37 C for 3 h. Cell viability was determined by the addition of MTT as described above. Proteolytic cleavages of PA and its mutant in vitro. PA and PAm were tested for susceptibility to the cleavage by trypsin and chymotrypsin. The proteins at 0.2 mg/ml were incubated with either trypsin (0.1 lg/ml) or chymotrypsin (0.5 lg/ml) for 20 min at room temperature in 25 mM Hepes, 1 mM CaCl2, and 0.5 mM EDTA, pH 7.5. Proteases were inactivated by adding 1 mM PMSF. Samples were boiled in SDS sample buffer to be electrophoresed and the results were confirmed further by Western blot. To verify the protection of the MAbs to proteolytic cleavage of PA by proteases, each MAb was incubated with PA for 1 h at room temperature prior to the adding of proteases. The molar ratio of MAb to PA is 2:1.

obtained were then characterized for antibody subclass. And the purified MAbs were compared their neutralizing activity against lethal toxin by toxin neutralization assay with rabbit anti-PA polyclonal antibody as a control. Upon the results, three MAbs (5E1, 2A8, and 5E12) were observed had TNA titer higher than polyclonal antibodies and the other MAbs. There are four domains in molecular structure of PA, each of which demonstrates different role in the toxin function [22]. We then determined the domains recognized by those MAbs with an ELISA detection in which we used a series of recombinant proteins comprising different domains of PA as coated protein, and confirmed the results by Western blot. It was found that four MAbs (5E1, 5E12, 2A8, and 1B3) bound to domain 2 of PA, one (4D10) bound to domain 3, and the other four (4B2, 4B6, 4F12, and 3E3) bound to domain 4. We further identified the relationship of those MAbs with the competitive binding assay, and found some MAbs recognized same epitopes. Table 1 lists the characteristics of the nine MAbs, including their immunoglobulin subclasses, TNA titer, domains of

Table 1 Characterization of toxin-neutralizing monoclonal antibodies Toxin-neutralizing MAb

Subclass

TNAtiterc

Binding Domain

5E12 2A8 5E1 1B3 4D10 4B2 4B6 4F12 3E3 RAba

IgG1 IgG2b IgG1 IgG2b IgG1 IgG2a IgG2a IgG1 IgG1 —b

12800 12800 6400 400 100 800 800 200 50 3200

Domain Domain Domain Domain Domain Domain Domain Domain Domain —

Potent neutralizing Epitoped 2 2 2 2 3 4 4 4 4

Epitope Epitope Epitope Epitope Epitope Epitope Epitope Epitope Epitope —

1 1 1 2 3 4 4 4 5

a

Rabbit anti-PA polyclonal antibodies. Not assayed. c The toxin-neutralizing activities of MAbs were assessed on a toxinsensitive cell line, as described at Materials and methods. The start concentration of each antibody was 1 mg/ml, and the antibody dilution that resulted in an optical density signal (A570) of 0.1 was used as a measure of the titer. d MAbs showed >50% of blocking in the competitive binding ELISA were considered recognize a same potential epitope. b

Results and discussion Production and identification of toxin-neutralizing MAbs About eighty mouse hybridoma clones specifically secreting anti-PA MAbs were generated by cell fusion technique and their ability to neutralize anthrax lethal toxin activities was screened by an in vitro toxin neutralization assay. Nine clones exhibiting toxin neutralization were developed further to get stable cell lines. Ascites from each hybridoma were produced in mice and MAbs were purified by Protein G chromatography. Toxin-neutralizing MAbs

Table 2 Phage immunization schedule and procedure Groupa

Immunogen

Route

Adjuvant

Doseb

1 2 3

Phage E22 Control phages Recombinant PA

i.p. i.p. s.c.

None None Freud’s adjuvantc

1011 pfu 1011 pfu 50 lg

a

Five female BALB/c (4–6 weeks old) mice per group. Immunization at 2-week intervals for three times. c The adjuvant of the first immunization is Freud’s adjuvant complete, the others are Freud’s adjuvant incomplete. b

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PA they bound to, and five potential neutralizing epitopes they recognized.

A

MW

97 kDa66 kDa-

Mapping of the epitope recognized by 5E1 and 2A8

PA(CH)

PA (T)

PA63 PA47 PA37

43 kDa-

We found three MAbs (5E1, 2A8, and 5E12) had stronger neutralizing activity than others and they all recognized a same epitope in the domain 2 of PA, which means there may be a dominant neutralizing epitope in this domain. So we tried to map this epitope by Phage display technique. We used two kinds of peptide library, the PhD-12 Library and the PhD-C7C Library to do the biopanning with 5E1 and 2A8 as the target respectively. After three rounds of biopanning, thirty clones binding 5E1 and fifteen clones binding 2A8 from PhD-12 Library and ten clones binding 5E1 from PhD-C7C Library were selected to confirm their binding with 5E1 and 2A8 by the competition experiment of ELISA. Phage clones with the blocking percent over 50% (16/30, 9/15, and 6/10, respectively) were sequenced their inserts. Analyzing the insert sequences of phage clones selected from two different peptide libraries, as shown in Table 3, we found a four amino acids consensus, SFFD, which was identical with the 312–315 residues of PA within the 2b2–2b3 loop of domain 2. There were several different residues among phage clones for the consensus, where the second residue F and the last residue D of SFFD were more conservative, while the first residue S and the third residue F were replaceable. The first residue S was irregularly replaced or absent in ten clones and the third residue F was occupied by W in seven clones, which may be explained by the speculation that 313-F and 315-D were key residues of the epitope bound by 5E1 and 2A8 which required to be verified. Among 31 phage clones being

PA

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31 kDa20 kDa-

PA20

14 kDa-

B 97 kDa66 kDa43 kDa-

PA

PA(CH)

PAm PAm(CH) PA (T) PAm (T)

PA63 PA47 PA37

31 kDa-

20 kDaFig. 1. Proteolytic cleavages of PA or PA mutant in vitro. Proteins at 0.2 mg/ml were incubated with either trypsin (T) (0.1 lg/ml) or chymotrypsin (CH) (0.5 lg/ml) for 20 min at room temperature in 25 mM Hepes, 1 mM CaCl2, and 0.5 mM EDTA, pH 7.5. Proteases were inactivated by adding 1 mM PMSF. Samples were separated by reduced SDS–PAGE 12% gels and stained with Coomassie blue. (A) Proteolytic cleavages of PA. Lane 1, molecular weight marker (MW); lane 2: PA; lane 3, chymotrypsin cleavage of PA (PA (CH)); and lane 4, trypsin cleavage of PA (PA (T)). (B) Proteolytic cleavages of PA mutant. Lane 1, PA; lane 2, chymotrypsin cleavage of PA (PA (CH)); lane 3, PA mutant (PAm); lane 4, chymotrypsin cleavage of PA mutant (PAm(CH)); lane 5, trypsin cleavage of PA (PA(T)); and lane 6, trypsin cleavage of PA mutant (PAm(T)).

Table 3 The consensus sequence of selected phage clones The insert sequences of phage clones binding 5E1 (E) or 2A8 (A) selected from the PhD-12 Peptide Library

The insert sequences of phage clones binding 5E1 selected from the PhD-C7C Peptide Library

E4- SSLPSFFDLPAR E5- TFFDNSKPPPSR E6-AFWDTFAFQSPD E7-AFWDTFAFQSPD E8-FFDKPTNAFFDL E10-TQYYSHFDLTRH E13-EFWDELAWRHPS E15-YPSFFDNPPLHA El7-QLHYPSFFDHIP E21-QDFHGTFWDTRP E22-SSSFFDANPKMSa E23-GSWWARSSFFDM E26-SPSSLFDIPLPA E28-YPSFFDNPPLHA E29-AFFDIPATTLPL E3O-WWDELPLGTPL

EC1- CSFFDRATC EC3-CLKGSFWDC EC5-CSFWDQLHC EC7-CLHRSFFDC EC8-CQSTFFDKC EC9-CMNSFWDKC

A1-TYHPNPVWSFFD A2-SSSFFDANPKMSa A5-ASFFDQITPNVD A6-SHFTQGSFFDLK A8-TSANSATFFDIL A12-SFFDLSSYDTAL A13-SSSFFDANPKMSa A14-TKNHFPAFFDLR A15-SSLCSFMDCGSL

Sequence of 2b2–2b3 loop of PA: . . .. . .302EVHGNAEVHASFFDIGGSVSAG323. . .. . . a

Identical phage clones binding 5E1 and 2A8.

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sequenced, only one (E30) had the insert different from the consensus sequence, which had a sequence of WWD that may represent a mimotope of PA. Proteolytic cleavages of PA and its mutant in vitro It was known that the sequence 312SFFD315 is related to the cleavage site of chymotrypsin [23]. Thus, we can confirm the relation of this sequence and MAbs 5E1, 2A8, and 5E12 via proteolytic assay. Chymotrypsin cleaved PA into an amino-terminal fragment of 37 kDa (PA37) and a carboxyl-terminal fragment of 47 kDa (PA47), while trypsin cleaved PA into an amino-terminal fragment of 20 kDa (PA20) and a carboxyl-terminal fragment of 63 kDa (PA63) (Fig. 1A). We constructed a mutant protein of PA (PAm) in which the sequence of 312SFFD315 was deleted. When added to the J774A.1 cells with LF, PAm was nontoxic even at 10 lg/ml, while the EC50 of wild type PA was about 10 ng/ml. When tested in proteolytic cleavage experiments, PAm was cleaved by trypsin, but not by chymotrypsin (Fig. 1B).In the Western blot experiments, three MAbs, 5E1, 5E12, and 2A8, recognized neither PA47 nor PA37, while rabbit anti-PA polyclonal antibodies (RAb) recognized both fragments and MAb 4B2 (which binds domain 4 of PA) only bound to PA47 (Fig. 2A); when PAm was tested, RAb and 4B2 bound to it, but 5E1 did not (Fig. 2B), and 5E12 and 2A8 only weakly bound the mutant (the data was not shown). Thus, 5E1 may recognize just the residues of 312SFFD315, while the A PA PA(CH)

PA PA(CH)

other two MAbs 5E12 and 2A8 may also recognize some residues nearby. To verify the protection of the MAbs to proteolytic cleavage of PA by proteases, the MAbs were incubated with PA prior to the adding of proteases. We found that when 5E1 and 5E12 beforehand incubated with PA, the cleavages were not done (Figs. 3A and B). This result showed that both MAbs protected PA against the cleavage of chymotrypsin. And 2A8 partially protected PA only leaving a small portion of PA cleaved into PA37 and PA47, while 4B2 did not protected PA form the cleavage completely (Figs. 3A and B). The results of proteolytic cleavage experiments provided more evidence that the epitope recognized by 5E1, 5E12, and 2A8 contains amino acids 312SFFD315. Immunization of phages contains the consensus sequence To assess whether the SFFD sequence was able to induce the epitope-specific antibodies in vivo, a phage clone contains the sequence (E22) was amplified and used as immunogen to inoculate mice, with recombinant PA and the non-specific phages as controls. After three times of immunization, serum samples were drawn from mice of different groups and anti-PA antibodies were detected. The results showed that mice immunized with E22 or recombinant PA generated anti-PA antibodies while mice immunized with the non-specific phages did not, as shown in Fig. 4. Although the phage containing the consensus insert

PA PA(CH)

PA PA(CH)

PA PA(CH)

PA47 PA37

5E1

5E12

B PA

PAm

5E1

2A8 PA

PAm

RAb

4B2 PA

RAb

PAm

4B2

Fig. 2. Western blot analysis of proteolytic cleavages of PA or PA mutant in vitro. (A) Proteolytic cleavages of PA. (B) Proteolytic cleavages of PA mutant The samples were separated by reduced SDS–PAGE 12% gels and then transferred onto nitrocellulose membranes. The membranes were then incubated with 5E1, 5E12, 2A8, 4B2 or rabbit anti-PA polyclonal antibodies (RAb) as the primary antibody and a HRP conjugated goat anti-mouse IgG (against the MAbs) or a HRP conjugated goat anti-rabbit IgG (against the RAb) as the secondary antibody. The bands were visualized by DAB substrate.

J. Zhang et al. / Biochemical and Biophysical Research Communications 341 (2006) 1164–1171

A

4B2 4B2+PA 2A8+PA 5E12+PA 5E1+PA PA PA (CH) (CH) (CH) (CH) (CH)

1169

MW

-97 kDa -66 kDa Hc PA47 PA37

-43 kDa -31 kDa

Lc -20 kDa -14 kDa

B

2A8+PA 5E12+PA 4B2+PA 5E1+PA (CH) (CH) (CH) (CH)

PA (CH)

PA

PA47 PA37

Fig. 3. Protective cleavages of protease experiments. (A) SDS–PAGE. The MAbs (5E1, 5E12, 2A8, and 4B2) were incubated with PA for 1 h at room temperature prior to the adding of proteases. The molar ratio of MAbs to PA is 2:1. The samples were separated by reduced SDS–PAGE 12% gels and stained with Coomassie blue. Lane 1, 4B2; lane 2, 4B2 incubated with PA prior to chymotrypsin cleavage; lane 3, 2A8 incubated with PA prior to chymotrypsin cleavage; lane 4, 5E12 incubated with PA prior to chymotrypsin cleavage; lane 5, 5E1 incubated with PA prior to chymotrypsin cleavage; lane 6, chymotrypsin cleavage of PA; lane 7, PA; and lane 8, molecular weight marker. Note. Hc, the heavy chain of an antibody; Lc, the light chain of an antibody. (B) Western blot analysis The samples were separated by reduced SDS–PAGE 12% gels and then transferred onto nitrocellulose membrane. The membrane was then incubated with RAb as the primary antibody and a HRP conjugated goat anti-rabbit IgG as the secondary antibody. The bands were visualized by DAB substrate. Lane 1, 2A8 incubated with PA prior to chymotrypsin cleavage; lane 2, 5E12 incubated with PA prior to chymotrypsin cleavage; lane 3, 4B2 incubated with PA prior to chymotrypsin cleavage; lane 4, 5E1 incubated with PA prior to chymotrypsin cleavage; lane 5, chymotrypsin cleavage of PA; and lane 6, PA.

could induce anti-PA antibodies, the titer of antibodies was much lower than PA immunization, and we did not observe the neutralizing activity of phage serum against lethal toxin in cytotoxicity assay. It may be explained by the weak immunogenecity of the epitope when the phage was used directly as immunogen. SFFD-epitope specific antibodies were present in anti-PA polyclonal antibodies We used two kinds of assays to testify the presence of SFFD-epitope specific antibodies in anti-PA polyclonal antibodies. In the competitive ELISA with PA as coated antigen, the anti-PA polyclonal antibodies from rabbits (RAb) could block the binding between PA and 5E1, and the percent of blocking was up to 58% at 10 lg/ml of

Fig. 4. ELISA of immunized sera from mice. The plates were coated with 2 lg/ml of recombinant PA and the sera from the mice immunized with phage 22, control phages or PA were diluted with 1:50. The HRPconjugated goat anti-mouse IgG (1:2000) was added and the plates were analyzed using a microplate reader at a wavelength of 450 nm. *Statistically significant differences (p < 0.01) vs. control.

RAb. In another ELISA with phages as coated antigen, RAb specifically bound to the phage clones containing SFFD sequence but did not bind to the non-specific phages (data not shown). So the SFFD-epitope specific antibodies were present in anti-PA polyclonal antibodies. These results further support that 312SFFD315 is a dominant epitope of PA. The current vaccine for anthrax has been licensed since 1970, known as anthrax vaccine adsorbed (AVA), which consists of a culture filtrate from an attenuated strain of B. anthracis adsorbed to aluminum salts as an adjuvant [1]. This vaccine is considered safe and effective, but is difficult to produce and is associated with complaints about reactogenicity among users of the vaccine [10,24]. It is known the bacterial capsule as well as anthrax toxin is virulence factors of B. anthracis. Among these toxin proteins, PA has become the major focus of work on vaccines and antibodies designed to treat or prevent anthrax infection since neither EF nor LF is capable of inducing cellular toxicity in its absence. It was known that antibodies to PA were crucial in the protection against anthrax toxin [11–15]. Recently, PA was also found to be associated with anthrax spores [17]. AntiPA antibodies-mediated, anti-spore activities may play a role in protection during the early stages of an anthrax infection [16]. In contrast, certain murine non-neutralizing monoclonal antibodies against PA have been shown to enhance the toxin mediated killing of macrophage cell lines [18,19]. So identification of antigenic epitopes in PA is important for development of the recombinant PA vaccine. On the other hand, many researches have been seeking to develop a vaccine or therapeutic agent that is safe and can be used before and after the exposure to virulent anthrax spores [10,24–26]. But being a natural toxin component, PA might be hazardous when used immediately following exposure. At present, it is underway through the use of dominant-negative inhibitory (DNI)

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mutants to replace PA in PA vaccines [27]. To obtain immunogenicity-enhancing DNI, the neutralizing epitopes of PA are required to be confirmed to direct the design of mutants. In early studies, the domain 4 of PA was known as containing the dominant neutralizing epitopes [28,29]. But recently, some epitopes or key residues of epitopes in other domains were also observed, especially in domain 2 [30– 32]. In this work, eight of nine neutralizing MAbs against PA bound to the epitopes in domain 2 or 4, which confirmed the proposal that both domin 2 and 4 have dominant neutralizing epitopes. We further found that three MAbs (5E1, 2A8, and 5E12) had stronger neutralizing activity against anthrax lethal toxin than others and they recognized a same epitope in the domain 2. And we mapped the epitope to the sequence 312SFFD315, in the 2b2–2b3 loop of PA. It was known that the 2b2–2b3 loop was involved in the transition of PA oligomers from prepore to pore on the membrane of target cells, which was a key step for anthrax toxin to enter the cells and exert its toxic effect [22,9]. Our results demonstrated for the first time that this loop also contains a dominant neutralizing epitope. The epitope identified in this study offers perspective for the development of a new epitope vaccine against anthrax. And the MAbs (5E1, 2A8, and5E12) to the 2b2–2b3 loop serve as interesting tools to study PA functional mechanism, as well as potential immunotherapeutic agents against anthrax toxin for their high toxin-neutralizing activity. Acknowledgments We thank Tianjing Lv, Shuling Liu for preparation and maintenance of the hybridomas. This work was supported by National Natural Science Foundation of China (30300016, 30571745). References [1] P.C. Turnbull, Anthrax vaccines: past, present and future, Vaccine 9 (1991) 533–539. [2] S.H. Leppla, Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells, Proc. Natl. Acad. Sci. USA 79 (1982) 3162–3166. [3] N.S. Duesbery, C.P. Webb, S.H. Leppla, V.M. Gordon, K.R. Klimpel, T.D. Copeland, N.G. Ahn, M.K. Oskarsson, K. Fukasawa, K.D. Paull, G.F. Vande Woude, Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor, Science 280 (1998) 734–737. [4] G. Vitale, R. Pellizzari, C. Recchi, G. Napolitani, M. Mock, C. Montecucco, Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages, Biochem. Biophys. Res. Commun. 248 (1998) 706–711. [5] M. Mourez, Anthrax toxins, Rev. Physiol. Biochem. Pharmacol. 152 (2004) 135–164. [6] P. Ascenzi, P. Visca, G. Ippolito, A. Spallarossa, M. Bolognesi, C. Montecucco, Anthrax toxin: a tripartite lethal combination, FEBS Lett. 531 (2002) 384–388.

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