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Keywords:

  • Recombinant protective antigen;
  • Bacillus brevis;
  • Bacillus anthracis;
  • Anthrax vaccine

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 μg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 °C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Bacillus anthracis secretes an exotoxin composed of three distinct proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). In animal test systems, these proteins interact in combinations that produce two distinct pathological presentations. When PA interacts with LF, it causes death in rats [1], but when it interacts with EF, it produces localized edema of the skin in guinea pigs and rabbits [2,3]. PA is thought to mediate the effects of EF and LF by binding to the receptors of sensitive eukaryotic cells [4]. After initial binding to a cellular receptor via its carboxy-terminal region [5], the amino-terminal part of PA is cleaved by a furin-like protease [6]. This process results in the release of a 20-kDa amino-terminal fragment and the heptamerization of a 63-kDa carboxy-terminal fragment [7,8]. The cleaved 63-kDa fragment of PA opens up the previously unavailable site to which LF or EF binds. The PA-LF or PA-EF complex that consequently forms then undergoes receptor-mediated endocytosis [9]. Acidification of the endocytic vesicles leads to the insertion of the 63-kDa PA fragment into the endosomal membrane and the translocation of EF or LF into the cytosol, where they exert a cytotoxic effect on the cell [10]. In addition to exotoxins, B. anthracis contains another virulence factor, capsule, which is composed of poly-γ-d-glutamic acid [11].

Protective antigen functions as a relevant antigen and as an immunogen, providing protection from anthrax infection in immunized animals [12,13]. Moreover, PA is an essential component of anthrax vaccine. Due to the low level of PA production in B. anthracis and the difficulty of separating it from other toxin components, research was initiated with the aim of inserting the PA gene into other bacterial species to produce greater quantities of PA. The PA genes have been cloned and expressed in Escherichia coli[14] and Bacillus subtilis[15,16]. However, the use of the former is associated with the risk of endotoxin contamination. On the other hand, the use of the latter has yielded PA in concentrations of up to 40 mg l−1 in culture supernatant. Thus, we developed a system for high production of PA using B. brevis 47-5Q based on a method developed by Udaka et al. [17]. Of the available host-vector systems for the production of foreign proteins in microorganisms, the use of a system based on B. brevis as a host, offers an advantage in which proteins are secreted directly into the culture supernatant, where they accumulate at high concentration in a relatively pure state. In addition, the secreted proteins are usually folded correctly and are thus soluble and biologically active [17]. The secretion vector, pNU212, is a derivative of pNU210 and is a high-copy-number expression vector that contains multiple promoters and the signal-peptide coding region of the extracellular middle-wall protein gene of B. brevis 47 [17]. In this paper, we describe a moderately efficient method, which produces rPA using this host-vector system and is also suitable for its use as a vaccine production system. The purified rPA was biologically active and elicited the production of a large amount of anti-PA antibodies and neutralizing antibodies in guinea pigs when formulated in either Rehydragel HPA or Ribi-Trimix. It completely protected immunized guinea pigs from a 100 LD50 challenge of B. anthracis spores.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Bacteria, plasmid and culture media

B. anthracis ATCC 14185 and ATCC 14578 were obtained from the American Tissue Culture Collection, and pT7-blue vector were obtained from Novagen. B. brevis 47-5Q was used as the host for overproduction and pNU212 was used as expression vector. The host strain and vector were kindly supplied by Yamagata [17]. B. brevis 47-5Q harbouring pNU212-PA was selected from T2U (polypeptone 1%, yeast extract 0.2%, meat extract 0.5%, glucose 1%, uracil 0.01%, pH 7.0) agar plates using 10 μg ml−1 erythromycin (EM). For the production of protein, it was grown at 30 °C in 5PY broth (glucose 2%, polypeptone-P 14%, yeast extract 0.5%, MgSO4· 7H2O 0.01%, FeSO4· 7H2O 0.001%, MnSO4· 4H2O 0.001%, ZnSO4· 7H2O 0.001%, and Uracil 0.01%, with the addition of 3% glucose after 2 days of incubation, pH 7.0) supplemented with 10 μg ml−1 erythromycin. The host strain, B. brevis 47-5Q, was cultured in the T2U medium. E. coli was grown in L broth and B. anthracis strains were grown in brain-heart infusion medium (Difco).

2.2Construction of an expression-secretion vector

Polymerase chain reaction (PCR) primers were synthesized at Bioneer Co. (Korea). The coding region of pagA was amplified by PCR from the ATCC 14185 DNA template using the oligonucleotide, 5′-ATTGGATCCGAAGTTAAACAGGAG-3′, which carries the sequences of the translational start site of pagA (position 1882 to 1905 according to the numbering system of Welkos et al. [18], containing the BamHI site (underlined)), and 5′-AGAGTCGACTTATCCTATCTCATAG-3′ (complementary to nucleotides 4083–4098, which are located downstream from the transcriptional terminator of pagA, containing the SalI site (underlined)). PCR was performed in a DNA thermal cycler (Perkin–Elmer) with a PCR reagent kit (Perkin–Elmer). Denaturation, annealing, and extension were performed at 95 °C for 1 min, 58 °C for 90 s, and 72 °C for 90 s, respectively. The reactions were repeated for a total of 30 cycles in a volume of 50 μl. The reaction mixture was run using electrophoresis on a 1.0% agarose gel. The PCR product was cloned into pT7-blue vector. Plasmids were digested with BamHI and SalI and then inserted between the BamHI and XhoI sites of pNU212 to generate pNU212-PA. To delete the extra nine nucleotides on the vector/insert junction sites of pNU212-PA, a QuikChange™ site-directed mutagenesis kit (Stratagene®) was used. As a result of site-directed mutagenesis, pNU212-mPA with no additional nucleotides was amplified from a pNU212-PA template using mutated primers. The forward primer was 5′-GTTGCTCCCATGGCTTTCGCTGAAGTTAAACAGGAGAACCGG-3′ and the reverse primer was 5′-CCGGTTCTCCTGTTTAACTTCAGCGAAACCATGGGAGCAACC-3′. PCR was carried out according to the instruction manual. Denaturation, annealing, and extension reactions were performed at 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 13 min, respectively. The reactions were repeated for 18 cycles in a volume of 50 μl. After PCR amplification, 1 μl of DpnI restriction enzyme (10 U ml−1) was added directly to each reaction and incubated at 37 °C for 1 h. DpnI-treated DNA was introduced into the host organism, B. brevis 47-5Q, using the tris-polyethylene glycol (Tris-PEG) transformation method [17]. Transformants were screened by restriction enzyme analysis using small-scale plasmid preparation. The deletion of nine nucleotides was confirmed by DNA sequence analysis.

2.3Screening of transformants

The plasmid pNU212-mPA was transformed into B. brevis 47-5Q using the tris-polyethylene glycol method [17]. B. brevis 47-5Q was grown at 37 °C for 4–5 h in 5 ml of T2U broth to an optical density at 660 nm of about 1.9. The following steps were performed at room temperature. Cells were collected by centrifugation at 8000g for 5 min, and the pellet was washed with 5 ml of 50 mM Tris–HCl (pH 7.5). The pellet was then suspended in 5 ml of 50 mM Tris–HCl (pH 8.5) and incubated for 60 min at 37 °C with gentle shaking. The cell pellet was harvested by centrifugation as described previously, washed with 1 ml of thiazoyl blue tetrazolium bromide solution (1.5 mg ml−1; MTP, Amresco), and uniformly suspended in 0.5 ml of MTP solution. pNU212-mPA dissolved in 50 μl MTP was added to the cell suspension and mixed well. Cells were collected by centrifugation, suspended in 1 ml of T2U broth containing 20 mM MgCl2, and incubated at 30 °C for 60 min. Erythromycin was then added to a final concentration of 0.1 μg ml−1 after incubation for 60 min. After 4 h incubation, 100 μl of the culture was spread on T2U agar containing 10 μg ml−1 erythromycin and the plates were incubated at 30 °C for 4 days. Transformants were then selected and grown on T2U broth containing erythromycin. Plasmids were isolated and the nucleotide sequences determined by automatic DNA sequencing.

2.4Nucleotide sequence of the inserted PA gene

The nucleotide sequence of the PA gene inserted into pNU212 was analyzed using a Big Dye™ Terminator Cycle sequencing kit (Perkin–Elmer) according to the manufacturer's instructions.

2.5Purification of PA

B. brevis 47-5Q (pNU212-mPA) was cultured in 1 liter of 5PY-EM at 30 °C for 4 days. The culture was harvested by centrifugation at 12,000g for 30 min at 4 °C. The culture supernatant was fractionated with 70% ammonium sulfate. After stirring for 1 h, the fractionate was centrifuged at 12,000g for 30 min at 4 °C. The ammonium sulfate pellet was dissolved in 20 mM Tris–HCl buffer (pH 8.0) and dialyzed against 4 ×2 l of the same buffer overnight. The dialyzed crude extract was centrifuged at 15,000g to remove insoluble material, and sterilized by filtration through a 0.22 μm disposable filter (Sartorius, Germany). A Hitrap-Q column (5 ml; Pharmacia, USA) was equilibrated with 25 ml of 20 mM Tris–HCl buffer (pH 8.0) at a flow rate of 0.5 ml min−1. Filtered crude extract was loaded onto the column, which was then washed with the same buffer. rPA was eluted using a NaCl gradient from 0 to 1 M in 20 mM Tris–HCl buffer (pH 8.0) and a flow rate of 0.5 ml min−1. Protein concentration was assessed from the optical density at 280 nm. All fractions were analyzed by SDS–PAGE and western blotting. Fractions containing the highest concentrations of rPA were pooled, concentrated using an ultrafiltration cell fitted with a PM10 membrane (Amicon, UK), and dialyzed overnight against 2 l of 20 mM Tris–HCl buffer (pH 8.0). Dialyzed rPA was filtered through a 0.22 μm filter and applied to a Hiload 16/60 Superdex 200 gel filtration column (Amersham Biosciences, USA) equilibrated with 20 mM Tris–HCl buffer (pH 8.0) and run at a flow rate of 0.5 ml min−1. Peak fractions were collected and analyzed for rPA as described previously. Fractions containing maximum rPA concentrations were pooled and concentrated to 10 mg ml−1. The concentrated rPA was equilibrated with 1.7 M (NH4)2SO4 in 20 mM Tris buffer (pH 8.0) and applied to a phenyl sepharose hydrophobic interaction chromatography column (Amersham Biosciences, USA). The rPA was eluted in a linear gradient of decreasing (NH4)2SO4 from 1.5 to 0 M at a flow rate of 0.5 ml min−1. The purified protein fraction was dialyzed against 20 mM Tris buffer (pH 8.0) and stored at −70 °C until use.

The purified rPA was subjected to 12% SDS–PAGE according to the method of Laemmli [19] and to immunoblot analysis as described by Towbin et al. [20]. Proteins were transferred onto a PVDF membrane (Bio-Rad) by electrophoresis for 40 min at 90 V. Detection and quantification of recombinant rPA were performed using an anti-PA monoclonal antibody (Abcam, UK) as the primary antibody, and goat anti-mouse IgG conjugated with alkaline phosphatase as the secondary antibody. Staining was performed using BCIP/NBT substrate.

2.6N-terminal amino acid sequence determination

The purified rPA was applied to SDS-PAGE electroblotted to a PVDF protein-sequencing membrane (Bio-Rad), and stained with Coomassie Brilliant Blue. The section of the membrane containing rPA was excised and applied to a protein sequencer system (ABI 477A/120A). N-terminal amino acid sequence analysis was performed at the Korean Basic Science Institute.

2.7Guinea pig immunization

Groups of female Hartley guinea pigs (Damul Science, Korea) weighing 300–320 g were immunized by intramuscular injection on days 0, 14, and 28 with 50 μg of the purified rPA. rPA was dissolved in 500 μl PBS containing Rehydragel HPA (alum hydroxide fluid gel, 250 μg; Reheis Inc., USA), or MPL-TDM-CWS adjuvant (monophosphoryl lipid A + trehalose dicorynomycolate + cell wall skeleton; Ribi-Trimix, Sigma) according to the manufacturer's protocol. Phosphate-buffered saline (PBS) was used as a negative control. Sera were obtained 2 days prior to immunization and 12 days after each vaccination.

2.8Quantification of antibodies to PA

Serum samples from individual guinea pigs were analyzed for PA-specific antibody responses using an enzyme-linked immunosorbent assay (ELISA). Pre-immune serum obtained 2 days before vaccination was used as a negative control. The wells of high-binding microplates (Costar, Corning Inc., USA) were coated with 0.1 μg of PA in 0.1 M carbonate buffer (pH 9.5) and overnight incubation at 4 °C. The wells were washed with PBS containing 0.05% (v/v) Tween 20 (PBST) and blocked with 3% (v/v) bovine serum albumin (BSA) in PBST. Serum samples were applied to the first column of each row and serially diluted with two-fold volumes of PBST until the last column of the row and incubated for 1 h at 37 °C. PA-specific antibodies were detected using a polyclonal goat anti-guinea pig IgG conjugated to peroxidase (Sigma). After washing with PBST, colour was developed using o-phenylenediamine (OPD) substrate. The reaction was stopped by adding 2N H2SO4, and the absorbance at 492 nm was measured using a microplate reader (TeCan Spectra Classic, Austria). The endpoint titers were defined as the reciprocal of the highest standard serum dilution that resulted in an absorbance three standard deviations greater than the average absorbance of negative control serum samples at the same dilution.

2.9In vitro toxin neutralization assay

Neutralizing antibody titers were determined according to the ability of serum samples to protect the mouse macrophage-like cell line, J774A.1, from the toxic effects of a cocktail of B. anthracis PA and LF. Cell viability was assessed according to the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) protocol described by Hansen et al. [21]. Monolayers of J774A.1 cells in 96-well flat-bottom tissue culture plates (SPL Plastic Labware, Korea) were incubated at 37 °C in DMEM cell culture medium containing 10% fetal bovine serum (FBS) up to a concentration of 4 ×105 cells per well. Before adding to the cells, guinea pig serum samples were diluted with cell culture medium containing 10% FBS, starting with a serum dilution of 1:10. rPA and LF (List Biological Laboratories Inc., USA) were added to all dilutions at final concentrations of 100 and 1000 ng ml−1, respectively. Serum dilutions were incubated with lethal toxin for 1 h at 37 °C. Medium was removed from the J774A.1 cell monolayers and 100 μl of the serum that had been incubated with lethal toxin was added to each well. Each sample was tested in duplicate. After 4 h of incubation at 37 °C under 5% CO2, 100 μl of MTT (3-[4,5-dimethylthylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; Sigma–Aldrich) was added to each well, resulting in a final concentration of 1.5 mg ml−1. After 1 h at 37 °C, the cells were lysed by adding 100 μl extraction buffer (90% isopropyl alcohol containing 25 mM HCl and 0.5% (w/v) SDS). Absorbance was then measured at 570 nm. To control for reproducibility, each assay included a dilution series of negative control serum. Four wells in each assay that received only lethal toxin served as blanks, and four wells containing only culture medium served as media controls. The neutralization titers were expressed as the reciprocal of the highest serum dilution exhibiting 0.1 absorbance units above the average absorbance of negative control serum samples at the same dilution.

The biological activity of purified rPA was assayed using the method described previously without anti-PA antibody fractions. The following LF concentrations were used: 0.01, 0.1, 1, 10 μg ml−1. rPA concentrations varied from 0.005 to 10 μg ml−1.

2.10Determination of the LD50

B. anthracis ATCC14578 spores were used for the in vivo protection experiment and to determine the LD50. Spores used for the challenge were prepared according to Ivins et al. [22] and resuspended in sterile PBS-0.1% gelatin. To determine the LD50 for the spore preparation used in this study, groups of female Hartley guinea pigs (weighing 300–320 g; Damul Science, Korea) were infected with doses of B. anthracis ATCC 14578 spores that ranged from 13 to 200 spores. Before injection, the spores were diluted in PBS with 0.1% gelatin and a dilution series was plated out on blood agar to determine the exact number of viable spores. Each guinea pig received 500 μl of the spore suspension, which was administered intramuscularly. Survival was monitored for 14 days.

2.11In vivo protection

To verify the protective activity of purified rPA in guinea pigs, five guinea pigs were vaccinated with 50 μg of rPA formulated in either Rehydragel HPA or Ribi Trimix as described previously. Fourteen days after the third immunization, the guinea pigs were challenged with 100 LD50 of B. anthracis ATCC14578 spores by intramuscular injection. After injection with B. anthracis spores, guinea pigs were observed for a period of 14 days. Animals surviving for 14 days after the challenge were considered survivors.

2.12Statistical analysis

The Mann–Whitney test was employed to assess the statistical significance of differences between independent sample groups. A two-sided probability value of <0.05 was considered statistically significant.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Construction of PA expression-secretion vectors in B. brevis 47-5Q

For the construction of pNU212-PA, the PA structural gene was amplified by PCR using primers containing the BamHI and SalI restriction enzyme sites, respectively. The 2.2-kb PCR product was subcloned into E. coli NovaBlue by the T7 vector. The PA gene of the plasmid cleaved with BamHI and SalI was inserted into BamHI and XhoI digested pNU212. The resulting pNU212-PA was introduced into B. brevis 47-5Q using the Tris-PEG transformation method. DNA sequencing of pNU212-PA was confirmed by the addition of nine nucleotides at the junction region of the vector; the PA insert and the deduced amino acid sequence were expected to have three additional amino acid residues on the N-terminal of PA. To produce recombinant PA with the same amino acid sequence as native PA, an additional nine nucleotides were removed by site-directed mutagenesis and the resulting pNU212-mPA was transformed into the host, B. brevis 47-5Q (Fig. 1).

image

Figure 1. Schematic diagram for the deletion of the three additional amino acids of rPA coded by pNU212-PA, which resulted in pNU212-mPA. The additional nine nucleotides were removed by site-directed mutagenesis. The nucleotide and amino acid sequences around the signal peptide cleavage site of mPA are shown at the upper right. N-terminal amino acid sequences of mPA produced by pNU212-mPA were the same as those of B. anthracis ATCC 14185.

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3.2DNA sequence of the inserted PA gene

The DNA sequence of the PA gene that was inserted into the plasmid pNU212 (pNU212-PA) was identical to that of B. anthracis ATCC 14185 except for the terminal BamHI restriction enzyme sites that were made for subcloning (data not shown). The deduced amino acid sequences were also the same as those of B. anthracis ATCC 14185 except for the additional three amino acids, Ala-Gly-Ser. After deleting the nine nucleotides, the resulting pNU212-mPA was found to have the same nucleotide sequence as authentic B. anthracis ATCC 14185. B. brevis 47-5Q carrying pNU212-mPA was used to produce the rPA.

3.3Production of PA in B. brevis 47-5Q

When B. brevis 47-5Q carrying pNU212-mPA was cultivated at 30 °C with shaking in 5PY-EM medium, rPA accumulation reached a maximum in 4 days (Fig. 2). The maximum concentration of rPA was approximately 300 μg ml−1. SDS–PAGE followed by staining with Coomassie Brilliant Blue also showed an 83-kDa band in gels made using samples of culture supernatant and cell lysates carrying pNU212-mPA. Immunoblotting of proteins extracted from the culture supernatant after SDS–PAGE demonstrated that specific reactivity existed between the 83-kDa PA band and the anti-PA monoclonal antibody (data not shown).

image

Figure 2. Time course of rPA production by B. brevis 47-5Q (pNU212-mPA). Time course of rPA production by B. brevis 47-5Q carrying pNU212-mPA during growth in 5PY medium over 5 days. Extracellular rPA was determined periodically by SDS–PAGE and immunoblot analysis (data not shown). Cell growth (optical density at 660 nm) and the total amount of protein were also monitored.

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3.4Purification of rPA

The crude supernatant was sterilized by filtration and then concentrated by 70% ammonium sulfate precipitation. The concentrate was dialyzed against 10 volumes of 20 mM Tris buffer and used for purification by fast protein liquid chromatography (FPLC). rPA was purified by anion exchange chromatography using a Hitrap Q column. rPA was eluted with approximately 0.1–0.15 M NaCl. Hitrap Q was effective in removing the outer cell wall and middle cell wall proteins from the culture supernatant. However, the breakdown product of rPA was also eluted with the full-sized rPA. Therefore, a further purification step was required. Partially purified pooled fractions containing rPA were applied to a Hiload 16/60 Superdex 200 gel filtration column and run at a flow rate of 0.5 ml min−1. rPA was eluted with a retention time of 120 min. Finally, a phenyl sepharose hydrophobic interaction chromatography column was used to produce higher purity rPA, which was eluted at the end of gradient, near to 0 M (NH4)2SO4. SDS–PAGE gel revealed an 83-kDa band, and western blotting using a monoclonal antibody detected some degradation of the rPA (Fig. 3). Typical yields were 70 mg of rPA from an initial culture supernatant volume of 1 l. The final purity was 90%.

image

Figure 3. SDS–PAGE profile of each purification step of rPA secreted by B. brevis 47-5Q (pNU212-mPA) using chromatography (a) or immunoblot analysis with anti-PA monoclonal antibody (b). Lane M, molecular weight marker; lane 1, culture supernatant; lane 2, concentrated culture supernatant with 70% ammonium sulfate precipitation; lane 3, after anion exchange (Hitrap Q column) chromatography; lane 4, after gel filtration (Hiload superdex 200) chromatography; lane 5, after phenyl sepharose interaction chromatography.

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3.5N-terminal amino acid sequence of rPA

The amino sequence of rPA from B. brevis 47-5Q carrying pNU212-mPA was Glu, Val, Lys, Gln, Glu, Asn, Arg, Leu, Leu, and Asn, which is identical to that of the native protective antigen of B. anthracis ATCC 14185 reported previously.

3.6Biological activity of rPA

To confirm that rPA produced from B. brevis is biologically active, rPA at various concentrations from 0.005–10 μg ml−1 was incubated with LF at concentrations of 0.01, 0.1, 1, 10 μg ml−1 on J774.1A cells. As expected, rPA plus LF exhibited cytotoxicity in J774A.1 cells in a concentration-dependent manner (Fig. 4). It has been reported that the amount of PA produced in B. anthracis Stern strain required to kill 50% of the J774A.1 with incubation of 40 ng ml−1 of LF was 8–9 ng ml−1[23]. In this study, the amount of rPA required to kill 50% of J774A.1 cells with incubation of 100 ng ml−1 of LF was 200 ng ml−1 (Fig. 4).

image

Figure 4. Biological activity of purified rPA on J774A.1 cells. Cells (4 ×105 cells ml−1) were cultured in 96-well culture plates in DMEM containing 10% FBS. LF at final concentrations of 0.01, 0.1, 1, or 10 μg ml−1 and the indicated final concentration of PA were added to the cultures. After 4 h at 37 °C, toxicity was measured using a MTT assay. Upper panels depict J774A.1 cells 4 h after addition of PA (1 μg ml−1) only (left) and after addition of PA (1 μg ml−1) plus LF (1 μg ml−1) (right). Magnification, 200×.

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3.7PA-specific antibody response of rPA in guinea pigs

To assess the humoral immune responses induced by purified rPA from the B. brevis host, we assayed serum from individual guinea pigs for PA-specific IgG. Groups of six guinea pigs were vaccinated intramuscularly on three occasions with 50 μg of rPA formulated with either Rehydragel HPA or Ribi-Trimix. Sera were obtained 12 days after each injection and anti-PA specific IgG was determined using the ELISA described previously. After three immunizations, rPA formulated in either adjuvant induced relatively high levels of serum anti-PA IgG. Booster injections significantly enhanced the IgG response (p < 0.05) (Table 1). Ribi-Trimix was four-fold more effective (p < 0.05) in enhancing the immunogenicity of rPA than Rehydragel HPA.

Table 1.  Vaccination against B. anthracis ATCC 14578
VaccinationaGroup antibody ELISA titerbSerum neutralization titercSurvivald (No. alive/5 tested)
123123
  1. aGuinea pigs were immunized by intramuscular injection on days 1, 14, and 28. Sera were obtained 12 days after each vaccination.

  2. bReciprocal geometric mean of anti-PA ELISA titers of sera.

  3. cReciprocal geometric mean of anti-PA neutralizing titers of sera.

  4. dGuinea pigs were challenged with 100 LD50 of spores of B. anthracis ATCC14578 at 14 days after the third immunization. Animals were observed for 14 days after spore challenges.

rPA + Rehydragel12515,91037,193502835665
rPA + Ribi25847,126146,99550226327715
PBS0000000

3.8In vitro neutralization

To evaluate the functionality of antibodies produced from vaccinations of rPA, sera from immunized guinea pigs were tested for their ability to neutralize PA by measuring the attenuation of lysis of J774A.1 cells in the presence of LF. rPA formulated in either Rehydragel HPA or Ribi-Trimix induced neutralizing antibodies, and booster injections enhanced neutralizing antibody titers (Table 1). After three immunizations, Ribi-Trimix induced a 4.9-fold higher (p < 0.05) level of neutralizing antibodies than Rehydragel HPA (Table 1). None of the negative control sera exhibited neutralization. In agreement with a previous report [24], Ribi-Trimix was more effective in inducing anti-PA and neutralizing antibodies than was Rehydragel HPA.

3.9Determination of the LD50

Five groups of guinea pigs were injected intramuscularly with 200, 100, 50, 25, or 13 B. anthracis 14578 spores. Survival was monitored during two weeks. The guinea pigs died in a dose-dependent manner (data not shown). The LD50, calculated according to Reed and Muench [25], was 74 spores.

3.10In vivo protection

To verify the effectiveness of the rPA from B. brevis as a vaccine candidate, we challenged immunized guinea pigs with 7400 spores (equivalent to 100 LD50) by intramuscular injection. Injections were administered 14 days after the third immunization. Without exception, control guinea pigs immunized with PBS died within 3 days (data not shown). In contrast, all guinea pigs immunized with rPA combined with adjuvants survived the lethal challenge (Table 1).

Anthrax is a bacterial disease caused by B. anthracis. The disease is normally associated with domestic livestock such as sheep, goats, and cattle. However, humans also become infected following exposure to infected animals or consumption of food produced from them [26]. Although natural infection of humans with anthrax is uncommon, recent events have highlighted the threat posed by the potential use of anthrax in terrorism and warfare. Currently available human vaccines based on the PA protein are effective [26–28], but they have limitations. The yield of PA in B. anthracis is very low and even highly purified PA was contaminated with small amounts of EF and LF, which may cause occasional mild side effects [28]. Therefore, many researchers have tried to express the PA gene in other bacterial systems such as E. coli[14], Baculovirus[29], Vaccinia virus[29], and B. subtilis[15,16]. Recently, a new candidate vaccine was constructed in which the capsule of B. anthracis, a poly-γ-d-glutamic acid, was conjugated to PA [30]. Protection against anthrax was enhanced by this vaccine [31].

Several animal models including guinea pigs [32], rhesus monkeys [33], and rabbits [34] were developed to study vaccine efficacy and to evaluate PA-based vaccine formulation or vaccination regimens. In this study, guinea pigs were chosen as an animal model for vaccination because guinea pigs are the most commonly used animal model in anthrax vaccine studies and were reported to demonstrate that anti-PA neutralizing antibody titers can be used as a surrogate marker for protection [35]. In addition, guinea pigs were used to determine the virulence of B. anthracis isolates in vivo and in testing the potency of newly manufactured lots of human cell-free anthrax vaccines prior to efficacy studies with larger and high-order species such as rabbits or nonhuman primates [36].

B. brevis is known to be an excellent host system for producing foreign proteins because it produces in abundance the desired protein and has little extracellular protease activity [17]. Another advantage is that the proteins are secreted directly into the culture supernatant, and accumulate in a relatively stable and pure state [17]. B. brevis secretes two major proteins in the two outer protein layers of the cell wall. During the early stationary phase of growth, cells continue to synthesize and secrete the cell wall proteins. These proteins do not stay on the cell surface but accumulate in the culture medium at concentrations of up to 20 g l−1, which is more than twice that achieved by intracellular proteins [17]. These characteristics of B. brevis were used by Udaka et al. [17] to develop an efficient host-vector system for the production of heterologous proteins. The expression-secreting vector, pNU212, contains the middle-wall protein (MWP) signal peptide-coding region, five tandem promoters, and dual translation initiation sites. The multicloning site on the plasmid is convenient for inserting foreign genes and for translational fusion with the 5′ terminal portion of mwp genes, forming a direct fusion with the MWP signal peptide. A PstI site located at the cleavage site of the MWP signal sequence can also be used for the production of foreign proteins with the correct NH2 terminus. However, a PstI site is located at base 2852–2857 of the PA gene of B. anthracis. We amplified the PA structural gene (at base 1891–4098) using the BamHI/SalI site containing PCR primers and cloned into the pT7 vector. After restriction digestion of pT7-PA, the insert was eluted and ligated with pNU212 digested with BamHI/XhoI. Transformant carrying pNU212-PA produced large amounts of PA in its culture supernatant, but the N-terminal sequence had three additional amino acid residues on the vector/insert junction site. Thus, site-directed mutagenesis using deleted primers on the junction site was performed and resulted in a clone carrying pNU212-mPA, which encoded the same nucleotide sequence as the native PA gene. We successfully expressed the PA gene in B. brevis 47-5Q and obtained much higher levels of rPA in the culture supernatant. The most efficient expression rate was 300 μg ml−1. In this experiment, we purified 70 mg of rPA from 1 l of culture supernatant of recombinant strain, B. brevis (pNU212-mPA). Moreover, the rPA exhibited biological activity in the cytotoxicity assay conducted using LF and J774A.1 cells. The antigenicity and immunogenicity of rPA as a vaccine component were confirmed by an anti-PA-specific ELISA and neutralization antibody tests. The immunization of guinea pigs with 50 μg of rPA combined with either Rehydragel HPA or Ribi-Trimix completely protected the guinea pigs from a 100 LD50 anthrax spore challenge. We anticipate that the rPA production system developed using B. brevis and pNU212-mPA will be useful for the development of a human anthrax vaccine.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

We thank J.S. Han, Y.Y. Chung, E.H. Kim, H.A. Chang, and K.T. Chung for their technical assistance. This work was supported by a Korea National Institute of Health grant (H.B.O.).

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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