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Infection and Immunity, April 2009, p. 1475-1482, Vol. 77, No. 4
0019-9567/09/$08.00+0     doi:10.1128/IAI.00828-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Anthrax Protective Antigen Delivered by Salmonella enterica Serovar Typhi Ty21a Protects Mice from a Lethal Anthrax Spore Challenge{triangledown} ,{dagger}

Manuel Osorio,1* Yanping Wu,1,{ddagger} Sunil Singh,1 Tod J. Merkel,2 Siba Bhattacharyya,1 Milan S. Blake,3 and Dennis J. Kopecko1

Laboratory of Enteric and Sexually Transmitted Diseases,1 Laboratory of Respiratory and Special Pathogens,2 Laboratory of Bacterial Polysaccharides, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland3

Received 3 July 2008/ Returned for modification 30 September 2008/ Accepted 18 January 2009


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ABSTRACT
 
Bacillus anthracis, the etiological agent of anthrax disease, is a proven weapon of bioterrorism. Currently, the only licensed vaccine against anthrax in the United States is AVA Biothrax, which, although efficacious, suffers from several limitations. This vaccine requires six injectable doses over 18 months to stimulate protective immunity, requires a cold chain for storage, and in many cases has been associated with adverse effects. In this study, we modified the B. anthracis protective antigen (PA) gene for optimal expression and stability, linked it to an inducible promoter for maximal expression in the host, and fused it to the secretion signal of the Escherichia coli alpha-hemolysin protein (HlyA) on a low-copy-number plasmid. This plasmid was introduced into the licensed typhoid vaccine strain, Salmonella enterica serovar Typhi strain Ty21a, and was found to be genetically stable. Immunization of mice with three vaccine doses elicited a strong PA-specific serum immunoglobulin G response with a geometric mean titer of 30,000 (range, 5,800 to 157,000) and lethal-toxin-neutralizing titers greater than 16,000. Vaccinated mice demonstrated 100% protection against a lethal intranasal challenge with aerosolized spores of B. anthracis 7702. The ultimate goal is a temperature-stable, safe, oral human vaccine against anthrax infection that can be self-administered in a few doses over a short period of time.


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INTRODUCTION
 
Bacillus anthracis, the etiologic agent of anthrax disease, is a gram-positive spore-forming bacterium (14). Anthrax is typically initiated by introduction of B. anthracis endospores into the host via the skin or gastrointestinal tract or through the respiratory epithelium following inhalation of airborne spores. Inhalational anthrax, the most severe form, is associated with rapid disease progression and death. The major virulence factors of B. anthracis are plasmid encoded. Plasmid pXO2 contains genes required for the synthesis of the antiphagocytic poly-{gamma}-D-glutamic acid capsule (15). Plasmid pXO1 encodes two binary exotoxins formed by combination of protective antigen (PA) with either lethal factor (LF) or edema factor (EF) (11, 25). PA forms homoheptameric structures that bind to protein receptors on the surface of eukaryotic target cells, leading to internalization of EF and LF (4). EF is a calcium- and calmodulin-dependent adenylate cyclase that increases the intracellular concentration of cyclic AMP. EF affects water homeostasis, thereby causing edema (18). LF is a zinc-dependent metalloprotease that cleaves members of the mitogen-activated protein kinase kinase family, preventing activation of p38 mitogen-activated protein kinase and thus inhibiting the activation of NF-{kappa}B-regulated genes, some of which are involved in antiapoptotic pathways in activated macrophages (28). When injected intravenously into small animals, lethal toxin (LeTx) (PA plus LF) is sufficient to cause death (26).

There is now considerable evidence that PA plays a major role in eliciting protective immunity against anthrax (24, 37) and that antibodies against PA are sufficient to protect against infection (1, 22). In fact, the only FDA-approved anthrax vaccine (AVA BioThrax) consists of an alum-adsorbed, formalin-treated culture supernatant of a toxigenic, nonencapsulated strain of B. anthracis whose primary component is PA, although small quantities of LF and EF are also present (39). The vaccine is administered subcutaneously in three doses given 2 weeks apart, followed by three injections at 6, 12, and 18 months and then yearly boosters. The route of immunization and dose schedule are based on an immunization regimen that was found to be effective in animal models (2).

In recent years, B. anthracis has attracted much attention because of its potential use as a biological weapon. However, anthrax remains endemic in many parts of the world, and every year many people die from environmental exposure to B. anthracis spores (40). The AVA BioThrax vaccine, although relatively safe and efficacious, suffers from several limitations; it requires multiple injectable doses over 18 months to stimulate protective immunity and in some cases has adverse effects, such as local pain, edema, and erythema at the injection site (17, 31, 35, 41). These limitations underline the need for new anthrax vaccine approaches, especially approaches that lead to the development of vaccines that can be easily administered (noninjectable), require fewer doses over a short time to induce protective immunity, and display markedly reduced reactogenicity.

Several approaches have recently been used to develop improved vaccines against anthrax. These approaches include the generation of purified recombinant PA without contaminants, PA conjugated with the poly-{gamma}-D-glutamic acid capsule, attenuated strains of B. anthracis, DNA vaccines, and PA expressed in alternative delivery systems, such as live attenuated viral and bacterial vectors (21). Here, we present data showing the feasibility of using the licensed, live, attenuated human typhoid vaccine strain Salmonella enterica serovar Typhi strain Ty21a as a vaccine vector for the production and delivery of recombinant anthrax PA to elicit a strong protective immune response against anthrax. Optimized expression of anthrax PA has resulted in a genetically stable oral vaccine candidate that efficiently secretes PA. Mice immunized with three doses of Ty21a secreting recombinant PA are completely protected from a lethal challenge with aerosolized spores of B. anthracis Sterne strain 7702.


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MATERIALS AND METHODS
 
Bacterial strains, plasmid vectors, and cell lines. S. enterica serovar Typhi strain Ty21a (galE ilvD viaB H2S–) (10) was used as the expression host for anthrax PA. Competent cells of Escherichia coli strains XL-1 Blue and XL10-Gold were purchased from Stratagene (San Diego, CA) and used for subcloning, plasmid amplification, and maintenance. Plasmid pGB-2, derived from pSC101, is a low-copy-number (five to seven copies/cell) cloning vector, which contains a selectable marker for streptomycin-spectinomycin resistance (3). pGB-2 was used for developing protein expression vectors in the Salmonella host. The wild-type B. anthracis PA gene was PCR amplified from plasmid pYS5, a kind gift from S. Leppla (NIH, Bethesda, MD). Plasmid WAM783 containing the E. coli HlyABD operon was a kind gift from R. Welch (University of Wisconsin, Madison). E. coli strains were grown aerobically in Luria-Bertani (LB) broth at 37°C with shaking (200 rpm) or on LB agar plates. Salmonella serovar Typhi Ty21a was grown in LB broth or tryptic soy broth or on tryptic soy agar supplemented with 0.01% galactose (Sigma-Aldrich, St. Louis, MO). Growth media were supplemented, as needed, with ampicillin (100 µg/ml), spectinomycin (100 µg/ml), or chloramphenicol (25 µg/ml). Mouse macrophage cell line RAW 264.7 (= ATCC TIB71) was used for determining anthrax toxin neutralization titers in mouse serum samples. RAW 264.7 cells were maintained at 37°C with 5% CO2 in supplemented Dulbecco modified Eagle medium (10% fetal bovine serum, 0.2 mM Glutamax, 100 U of penicillin/ml, 100 µg/ml streptomycin) (Invitrogen, Carlsbad, CA).

Synthetic PA gene for optimal expression and stability. A synthetic gene encoding an optimized PA protein (PAop) was created by modifying the codon sequence of the wild-type PA gene for optimal expression in gram-negative bacteria. A total of 148 codons were modified for optimal codon utilization, which resulted in no change in the amino acid sequence of the encoded protein. Separately, the resistance of PA to proteolytic degradation was enhanced by replacing the furin cleavage site RKKR167 with SNKE167 and by eliminating a chymotrypsin cleavage site via deletion of FF314 and a substitution at position 308, E308D (32, 36) (see the supplemental material for sequence details).

Recombinant expression vector construction. (i) Generation of lpp, htrA, and nirB promoter regions and fusion to genes encoding PA. The htrA promoter region was PCR amplified from S. enterica serovar Typhi strain Ty21a genomic DNA using 5' and 3' end primers containing HindIII and NdeI restriction sites, respectively. The 3' NdeI primer includes a strong Shine-Dalgarno sequence (AGGAG) cloned just upstream of the PA ATG codon. The optimized synthetic gene encoding mature PA was used for expression in the Ty21a host. This gene was flanked by an NdeI restriction site at the start codon and by a BamHI site after the stop codon. Both the htrA promoter region and the gene encoding PAop were cloned (three-way ligation) into the HindIII and BamHI restriction sites of plasmid pGB-2 to create plasmid pHtrA-PAop. A synthetic nirB promoter was created by Taq polymerase extension of two 55-bp annealed oligonucleotides containing a complementary 20-bp region at their 3' ends. The 90-bp product was then PCR amplified using oligonucleotide primers introducing HindIII (5') and NdeI (3') restriction sites. This product, together with the PAop gene with sticky NdeI (5') and BamHI (3') ends, was then cloned into the HindIII and BamHI restriction sites of plasmid pGB-2, generating plasmid pNirB-PAop. An optimized lpp promoter (13) and an enhanced Shine-Dalgarno sequence were added on to the 5' end of the gene encoding PAop by sequential extension of this gene via two successive PCR amplifications that resulted in a complete lpp promoter fused to the PAop gene. This product was then cloned into the HindIII and BamHI sites of pGB-2, generating plasmid pLpp-PAop. The resulting recombinant products were initially transformed into E. coli XL-1 Blue for amplification and maintenance.

(ii) Construction of plasmid vectors for production of secreted PA. For secretion of PA from the Salmonella host, genetic fusions were created between the gene encoding PA and the DNA region encoding the C-terminal signal sequence (60 amino acids) of the HlyA protein. The region encoding the E. coli HlyA signal sequence (hlyAs) and the complete genetic regions encoding the secretion effectors HlyB and HlyD (4.0-kb fragment) were isolated from plasmid WAM783 by digestion with the NsiI and BamHI restriction enzymes. The gene encoding PA in plasmids pHtrA-PAop, pNirB-PAop, and pLpp-PAop was replaced with a gene lacking the stop codon and instead containing an NsiI restriction site upstream of the C-terminal BamHI site. Deletion of the stop codon and introduction of the NsiI site, performed by PCR amplification, allowed in-frame ligation to the hlyAs-hlyBD cassette isolated from plasmid WAM783. The secretion cassette (hlyAs-hlyBD) was introduced into the new pHtrA-PAop and pNirB-PAop constructs at the NsiI and BamHI sites, creating recombinant vectors pHtrA-PAop-hlyAs, pNirB-PAop-hlyAs, and pLpp-PAop-hlyAs. The resulting plasmids were first transformed into E. coli XL-10 competent cells for amplification and storage. Successful manipulation and generation of recombinant plasmids were confirmed by restriction enzyme analysis and DNA sequencing. For protein expression analysis, all recombinant plasmids were transferred, via electroporation using a GenePulser II electroporator (Bio-Rad, Hercules, CA), into Salmonella serovar Typhi Ty21a.

Plasmid stability. The stability of pGB-2-based vaccine constructs in Ty21a was determined as described previously (44). Plasmid vectors expressing PA under control of the htrA, nirB, or lpp promoter were evaluated for maintenance in the Ty21a host. Overnight cultures of the vaccine constructs, grown on LB medium containing 0.01% galactose and 100 µg/ml spectinomycin, were diluted to obtain a concentration of approximately 100 CFU per ml in fresh medium lacking spectinomycin. The diluted cultures were then grown overnight for ~25 generations with agitation at 37°C. The overnight cultures were diluted again to obtain a concentration of 100 CFU per ml in fresh nonselective LB medium and grown again overnight, for a total of 50 generations. Aliquots were removed from the cultures after one and two nights of nonselective growth, plated onto LB agar without spectinomycin, and incubated at 37°C. Approximately 100 colonies of each strain were assessed for plasmid maintenance after 25 or 50 generations by evaluating their ability to grow on LB agar containing spectinomycin. Maintenance of PA expression was also determined by colony immunoblotting using a PA-specific rabbit polyclonal antibody.

Characterization of PA expression. Salmonella serovar Typhi Ty21a derivatives carrying a plasmid vector for PA production were grown overnight in LB medium containing 100 µg/ml spectinomycin at 37°C in a shaking incubator. Each overnight culture was then diluted 1:20 in fresh LB medium (without antibiotics) and grown to an optical density of 0.8 (~4 h). The bacterial cultures were then harvested by centrifugation, and the supernatants were removed. Both supernatant and pellet fractions were processed immediately or stored at –80°C for future use. Each supernatant fraction was concentrated 10-fold by centrifugation using a Vivaspin 6 concentrator (Vivascience, Sartorious Corp., Edgewood, NY) or by trichloroacetic acid (TCA) precipitation. TCA precipitation was performed by addition of an equal volume of 20% TCA, followed by incubation of the sample on ice for 1 h and centrifugation at 4°C for 30 min. The resulting TCA pellet was washed once with 1 volume of cold acetone and finally dissolved in 1/10 the original volume in 10 mM Tris-HCl (pH 8.0). The bacterial pellets were lysed using a ReadyPreps protein preparation kit (Epicentre Biotechnologies, Madison, WI), and the final lysate volume was adjusted so that it was equal to the concentrated supernatant volume. For in vitro induction of PA expression, the Ty21a derivatives carrying the plasmid constructs under htrA promoter control were grown at 43°C, and the derivatives carrying the plasmid constructs under control of the nirB promoter were grown at 37°C under reduced oxygen conditions (CO2 chamber) with shaking. Proteins were separated by electrophoresis using NuPAGE 4 to 12% bis-Tris gels (Invitrogen, Carlsbad, CA). A supernatant or pellet fraction equivalent to approximately 0.3 optical density unit was loaded in each lane. As a control, purified recombinant PA (50 to 150 ng) was loaded in a lane. Proteins were visualized by staining with Coomassie blue or by transfer to phenylmethylsulfonyl fluoride membranes for immunoblotting. The membranes were blocked with 4% BLOTTO (Santa Cruz Biotechnology, Santa Cruz, CA) and probed with a rabbit polyclonal serum raised against PA. The protein standard used in these assays is MagicMark XP from Invitrogen (Carlsbad, CA). PA concentrations were determined by densitometric analysis using purified recombinant PA as a reference.

Mice and immunization. Female 4- to 6-week-old A/J mice were purchased from the National Cancer Institute Division of Cancer Treatment (Frederick, MD) or Taconic (Germantown, NY). All animal experiments were performed according to the guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Bethesda, MD). Animals were maintained in the Small Animal Facility of the Center for Biologics Evaluation and Research of the U.S. Food and Drug Administration (Bethesda, MD) and were used with the approval of the FDA's Institutional Animal Care and Use Committee. Mice were immunized via the intranasal (i.n.) or intraperitoneal (i.p.) route with three biweekly doses containing Salmonella serovar Typhi Ty21a producing PA under control of the htrA or nirB promoter with or without the Hly secretion machinery. Control mice received three doses of Ty21a alone. Mice that were immunized i.n. received 5 x 108 CFU per dose, and mice that were immunized i.p. received 5 x 107 CFU per dose. i.n. immunization was performed by administering 20 µl of a bacterial solution to the nares. Mice were first lightly anesthetized by i.p. injection of a solution consisting of ketamine and xylazine. i.p. immunizations were administered by injection of a 0.5-ml bacterial suspension.

Total circulating anti-PA IgG antibody titers. Total serum immunoglobulin G (IgG) antibody titers to PA were determined using a quantitative anti-PA enzyme-linked immunosorbent assay (ELISA) method as previously described by us (27) and other workers (34, 45). Briefly, 96-well microtiter plates (Immunolon 2HB; ThermoLabsystems, Franklin, MA) were coated with 100 µl/well of recombinant PA (1 µg/ml) overnight at 4°C. The plates were then washed with phosphate-buffered saline (PBS) plus 0.05% Tween and blocked with 3% bovine serum albumin in PBS for 1 h at 37°C. The plates were incubated with 100 µl of serially diluted (1:100 to 1:300,000) serum samples at 37°C for 1 h. The plates were then incubated for 30 min at room temperature with purified horseradish peroxidase-conjugated goat anti-mouse IgG (KPL, Gaithersburg, MD) diluted 1:1,000 in blocking buffer. Finally, the plates were incubated for 15 to 20 min at room temperature with 100 µl of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate (KPL, Gaithersburg, MD). The reaction was stopped by adding 100 µl of ABTS peroxidase stop solution (KPL, Gaithersburg, MD). Values for absorbance at 405 nm were obtained using a Molecular Devices (Sunnyvale, CA) VERSAmax microplate reader. Samples were assayed in triplicate, and the endpoint antibody titer was defined as the maximum dilution giving an absorbance at 405 nm of more than 0.2. The results are expressed below as the reciprocal of the dilution multiplied by the absorbance value.

Toxin-neutralizing titers. Serum samples from each mouse group were randomly paired to reduce the number of samples by one-half and to ensure that enough serum was available to perform each of the toxin neutralization assays. The paired serum samples were tested for the ability to prevent lysis of RAW 264.7 murine macrophages in the presence of anthrax LeTx. Toxin neutralization assays were performed as previously described (30), except that RAW 264.7 cells were used instead of J774.1 cells. Briefly, serial dilutions (1:300 to 1:300,000) of the serum samples were prepared in triplicate in a 96-well microtiter plate (Corning Costar, Lowell, MA) with supplemented RPMI medium containing LeTx (50 ng/ml PA and 40 ng/ml LF) and incubated for 1 h at 37°C. A plate-to-plate transfer was then done from the titration plate to 96-well plates containing a monolayer of RAW 264.7 cells. The RAW 264.7 cells had been plated the day before at a density of 5 x 105 cells per ml. The plates were then incubated in a CO2 (5%) incubator at 37°C for 4 h. At the end of the incubation period, 25 µl of 3-(4,5-dimethylhyazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/ml in PBS; Sigma-Aldrich, St. Louis, MO) was added to each well. The plates were incubated at 37°C for an additional 2 h, and subsequently the contents were lysed and the precipitate was dissolved by adding 100 µl per well of solubilization buffer containing 20% (wt/vol) sodium dodecyl sulfate in 50% dimethylformamide (pH 4.7). The optical density at 570 nm was determined using a reference reading at 690 nm with a Molecular Devices VERSAmax microplate reader. Cell viability (expressed as a percentage) was calculated by using the ratio of the corrected optical density of the wells exposed to toxin to the corrected optical density of the control wells (no toxin added). Cell viability was then plotted against the serum dilutions, and the toxin-neutralizing titer was estimated by nonlinear regression analysis using the reciprocal of the serum dilution resulting in 50% cell viability. Data analysis was performed with the GraphPad Prism version 5 software package.

Challenge of mice with spores of toxigenic, nonencapsulated B. anthracis strain 7702. Mice were exposed for 90 min to aerosolized spores (5 x 109 spores per ml in deionized water with 0.01% Tween 80) prepared from B. anthracis strain 7702(pXO1+, pXO2–), as described elsewhere (29). The spore aerosol was generated using a six-jet Collison nebulizer equipped with a precious fluid jar containing a 10-ml inoculum (BGI Incorporated, Waltham MA). The mice were exposed using a nose-only exposure system (CH Technologies, Westwood, NJ). Prior to exposure, mice were supplied with fresh air for 10 min to allow the respiratory rate to normalize. Spores were prepared as described previously (29). Mice were monitored for survival for 20 days.

Statistical analysis. Statistical analyses were performed by means of an unpaired t test using GraphPad Prism version 5. A P value of ≤0.05 (two tailed) was considered to be statistically significant.


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RESULTS
 
Plasmid construction and analysis of PA expression and secretion. Our aim was to create genetically stable plasmid expression systems for optimized expression of anthrax PA in S. Typhi vaccine strain Ty21a. For this purpose, we used a synthetic gene encoding an optimized PA protein (PAop). The PAop gene consists of modified codons for optimal expression in gram-negative bacteria and has mutations to eliminate proteolytic cleavage sites, such as furin and chymotrypsin sites, that enhance the stability of the secreted protein in the mammalian host (32). Our initial studies showed that use of this synthetic gene resulted in a >10-fold increase in PA expression compared to the expression in recombinant clones containing the unmodified PA gene (data not shown). Other optimizing components of the PA expression systems included environmentally regulated promoters, such as the nirB and htrA promoters, which are only weakly induced during broth growth but are known to be fully activated in mammalian host cells (5). In addition, an optimized lpp promoter was used for constitutive expression (13). The low-copy-number plasmid pGB-2, which has been shown to be stably maintained in Ty21a (44), was used as the backbone for plasmid construction.

The PAop gene was cloned downstream from the lpp, nirB, or htrA promoter in plasmid pGB-2 (Fig. 1A). For extracellular expression of PA, an in-frame genetic fusion of the PAop gene, lacking a stop codon, was created with the DNA region encoding the last 66 amino acids (i.e., the secretion signal peptide) of the E. coli alpha-hemolysin protein, HlyA (Fig. 1A). This plasmid vector also carries the genes encoding HlyB and HlyD, which are necessary components of the transport apparatus for HlyA and fusion proteins containing the C-terminal HlyA secretion signal (9).


Figure 1
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  		FIG. 1. (A) Schematic representation of plasmid expression systems for anthrax PA. P indicates an inducible or constitutive promoter (htrA, nirB, or lpp). To secrete PA extracellularly, the PAop gene was fused to the HlyA secretion signal sequence and linked to the HlyBD secretion effectors. (B) Immunoblots of pellet (lanes P) and supernatant (lanes S) fractions derived from S. Typhi Ty21a cells expressing PA and control Ty21a pellet fractions. Lanes rPA contained approximately 50 to 100 ng of purified recombinant PA and were used as size markers. Both lysed pellet and supernatant fractions were normalized before loading, and approximately 0.2 optical density at 600 nm unit was loaded per lane. The presence of PA was detected by probing with a rabbit polyclonal antibody against PA. Lane M contained a commercial protein size ladder (MagicMark XP from Invitrogen), which was designed to be visualized directly on Western blots as each marker contains an IgG binding site.

For analysis of PA production in Salmonella strain Ty21a, derivatives either expressing PA in the intracellular compartment or secreting it into the culture medium were grown without antibiotic selection to logarithmic phase over a 4-h induction period and subsequently harvested by centrifugation. The supernatant fractions were then concentrated approximately 10-fold. The pellet fractions were lysed in a volume equal to that of the concentrated supernatant fractions. Equal volumes of concentrated supernatant and lysed pellet fractions were analyzed by Western blotting using a polyclonal rabbit serum raised against PA. As shown in Fig. 1B, the majority of PA produced by a Ty21a construct carrying the gene encoding PAop without the HlyA secretion signal was detected in the pellet fraction. In contrast, PA fused to the HlyA secretion signal is efficiently exported to the culture medium, as most of the protein is found in the supernatant fraction. Based on the translated DNA sequence, the predicted molecular mass of recombinant PA is approximately 83 kDa, the predicted molecular mass of nonsecreted PAop is about 82 kDa, and the molecular mass of the secreted PAop should be approximately 89 kDa. Therefore, the observed higher molecular mass of PA secreted from the Salmonella host is likely a reflection of the additional 66 amino acids that constitute the secretion signal derived from the hemolysin A protein, as this secretion signal is not known to be cleaved (6, 12, 16). Semiquantitative analysis of Western blots for induced and lysed Ty21a PAop constructs showed that the amount of PA produced was ~18 µg per 6 x109 cells (data not shown). Although the PA protein expressed in the Ty21a host has been engineered to possess enhanced protease resistance, the additional lower-molecular-weight bands in the Western blots (Fig. 1B) are probably due to the activity of bacterial proteases.

Plasmid stability in the Ty21a vector. Recombinant plasmids utilizing either the lpp, htrA, or nirB promoter and carrying the genes encoding either PAop or PAop fused to the HlyA secretion signal were introduced into S. Typhi strain Ty21a. The genetic stability of the expression plasmids in the resulting Ty21a clones was analyzed by monitoring growth in the absence of antibiotic selection for approximately 25 generations (12 h) and 50 generations (24 h). Plasmid maintenance in the resulting colonies was determined both by examining resistance to spectinomycin and by examining PA expression using colony immunoblotting with an anti-PA polyclonal serum. The expression plasmid producing PA in the cytoplasmic space was highly stable, with 100% retention in Salmonella after 25 generations and 98% retention after 50 generations in the absence of selective pressure (Table 1). Inclusion of the Hly secretion components, HlyB and HlyD, in nirB- or htrA-regulated expression plasmids resulted in slightly lower, but still significant, stability of the plasmids, with 85 to 98% retention after 25 generations (Table 1). The presence of the lpp constitutive promoter resulted in a significant decrease in plasmid stability, as only 5% of the bacterial cells expressed PA after 25 generations of growth in the absence of antibiotic selection (Table 1).


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  		TABLE 1. Stability of plasmid-based PA expression in recombinant Ty21a strains

Immunogenicity of vaccine constructs in A/J mice. Ultimately, the Ty21a vector secreting PA must be assessed for its ability to protect immunized animals against an i.n. challenge with aerosolized spores of B. anthracis, a challenge that may more closely resemble actual human respiratory exposure. A/J mice are known to be susceptible to i.n. challenge with aerosolized spores of B. anthracis strain 7702(pXO1+, pXO2–) (43). Therefore, the abilities of the different PA-producing Ty21a constructs to induce a protective immune response were first examined by using i.n. or i.p. immunization of A/J mice. Groups of 10 4- to 6-week-old female A/J mice were immunized three times, 2 weeks apart, with Ty21a expressing secreted PA under control of either the nirB or htrA promoter and with or without a fused extracellular secretion signal. Due to the instability of the expression plasmid containing the lpp promoter (Table 1), these constructs were not included in the immunogenicity studies. The mice immunized via the i.p. route received 5 x 107 CFU per dose, whereas those immunized i.n. received 5 x 108 CFU of vaccine construct. Control mice were immunized i.p. or i.n. with Ty21a carrying the plasmid vector, pGB-2, lacking the PA gene. Serum samples were obtained from vaccinated mice 2 weeks after the last immunization, and the PA-specific IgG titers were analyzed by ELISA. Total circulating PA-specific IgG titers are shown in Fig. 2. Immunization via the i.p. route with the HtrA-PAop construct (not secreting PA) resulted in a peak IgG anti-PA geometric mean titer (GMT) of 600. In striking contrast, immunization with a construct secreting PA (HtrA-PAop-hlyAs) elicited the highest titers, with a GMT of 30,000 (IgG titer range, 5,800 to 157,000) for PA-specific antibodies. Immunization of mice via the i.n. route with the HtrA-PAop-hlyAs construct resulted in a significantly lower serum anti-PA GMT (3,900). Compared to the HtrA-PAop-hlyAs derivative, the construct expressing secreted PA under control of the nirB promoter and given i.p. was not as effective in eliciting a strong response against PA, generating only a modest titer (GMT, 3,800). Control strain Ty21a(pGB-2) did not elicit PA-specific antibody titers in vaccinated animals.


Figure 2
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  		FIG. 2. Immunogenicities of vaccine constructs in A/J mice. Mice were immunized via the i.n. or i.p. route with three biweekly doses containing Salmonella serovar Typhi Ty21a producing PA under control of the htrA or nirB promoter. i.n. immunized mice received 5 x 108 CFU per dose, and mice immunized i.p. received 5 x 107 CFU per dose. Sera were obtained 2 weeks after the last immunization, and circulating, PA-specific IgG antibody titers were measured by ELISA.

Anthrax LeTx neutralization by sera from immunized mice. To evaluate the protective quality of the immune response in mice vaccinated with the Ty21a constructs producing PA, we investigated the ability of immune serum to neutralize the cytotoxic activity of anthrax LeTx. Serial dilutions of serum samples, prepared as described in Materials and Methods, were assessed for the ability to protect cultured murine RAW 264.7 cells from killing when they were incubated with a lethal dose of LeTx (50 ng/ml PA and 40 ng/ml LF). RAW 264.7 cells were challenged with LeTX previously incubated with serial dilutions (1:300 to 1:300,000) of serum samples from vaccinated mice. The LeTx neutralization curves for pooled sera from each of the vaccinated groups are shown in Fig. 3. The serum dilution resulting in protection of 50% of the RAW 264.7 cells was defined as the neutralizing titer. The estimated neutralizing titers are summarized in Table 2. i.p. immunization again resulted in a more efficient immune response (neutralizing titer, 16,241) than i.n. immunization with the same construct. Interestingly, although NirB-PAop-hlyAs given i.p. and HtrA-PAop-hlyAs given i.n. elicited similar total IgG titers to PA, the i.p. route induced slightly higher titers of toxin-neutralizing antibodies. However, statistical analysis showed that the difference was not significant (P = 0.51). No detectable neutralizing titers were observed in mice immunized with Ty21a(pGB-2) not expressing PA, even at a 1:300 dilution (Fig. 3).


Figure 3
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  		FIG. 3. Anthrax LeTX neutralization curves. Serial dilutions of sera collected from A/J mice immunized as described in the legend to Fig. 2 were tested for the ability to protect cultured RAW 264.7 cells from the cytotoxic effects of anthrax LeTx. Serum samples represent sera of randomly paired samples for each group of 8 to 10 mice. Protection is expressed as the percentage of viability for the RAW 264.7 cells compared to a control lacking LeTx. LeTx-neutralizing titers were estimated by nonlinear regression analysis to determine the serum dilution that results in 50% RAW 264.7 cell viability. The error bars indicate the ranges of values at the serum dilutions.


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  		TABLE 2. Total anti-PA IgG titers and toxin-neutralizing titers in A/J mice immunized with Ty21a PA vaccine constructs

Protection from aerosolized anthrax spore challenge. Mice immunized every 2 weeks with S. Typhi strain Ty21a secreting PA or a Ty21a(pGB-2) vector control and given a total of three doses were challenged 2 weeks after the last immunization with 4 x 106 CFU (~20 50% lethal doses [LD50]) of aerosolized spores of B. anthracis strain 7702 as described previously (23). Following challenge, mice were monitored for survival for 20 days. As expected, mice immunized with the Ty21a(pGB-2) vector control alone were not protected, and all mice died between days 3 and 5 (Fig. 4). Mice immunized with the vaccine construct HtrA-PAop producing nonsecreted PA were only partially protected, and 60% of these animals succumbed to the disease. In contrast, mice immunized with any of the optimized expression constructs secreting PA were completely protected from this lethal aerosol spore challenge. This challenge study was repeated, and virtually identical results were obtained.


Figure 4
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  		FIG. 4. Challenge with aerosolized B. anthracis strain 7702 spores. Mice immunized as described in the legend to Fig. 2 were challenged i.n. with 4 x 106 CFU (~20 LD50) of aerosolized spores. The challenged mice were monitored for 20 days.


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DISCUSSION
 
Attenuated strains of S. enterica provide a well-studied vehicle for oral presentation of heterologous antigens to stimulate mucosal, humoral, and cell-mediated immunity (for a review, see reference 20). Attempts to deliver many bacterial and viral antigens (including antigens from Shigella, E. coli, Vibrio cholerae, Francisella tularensis, Clostridium tetani, streptococci, dengue virus, hepatitis B virus, human papillomavirus, influenza virus, and measles virus) via attenuated Salmonella vectors have been made (20). An important advantage of live attenuated oral delivery systems for a rapid response for public health use and emergencies is the ability to circumvent the need for syringes and needles or for administration by a trained health care professional.

There is already significant evidence demonstrating that an anti-PA immune response can protect against aerosolized anthrax spore exposure. However, the existing PA-based injectable vaccine has been associated with adverse reactions, and the recommended immunization regimen requires six or more doses over a period of 18 months. Our goal has been to develop an oral vaccine for human use that can be rapidly distributed and self-administered in a few doses over a short time period and that protects against anthrax infection. Because of the impressive safety record and long protection induced by the licensed oral typhoid vaccine, we used S. Typhi strain Ty21a as the delivery platform for PA. The plasmid constructs used included a codon-optimized synthetic B. anthracis PA gene, which also lacked two proteolytic cleavage sites, thus providing enhanced resistance to proteolytic degradation of the PA protein produced (32). Use of this enhanced PA gene resulted in expression of PA that was greater (10-fold) than that obtained with the wild-type PA gene. In vivo-inducible promoters, such as the htrA and nirB promoters (5), were employed to maximize PA production in the target host while low expression was maintained during broth growth, thus enhancing strain stability during vaccine manufacture. Genetic fusions of the synthetic PA gene to the C terminus of HlyA were generated to allow extracellular secretion of PA (Fig. 1A) (9). The expression plasmids containing inducible promoters displayed greater stability than the constitutive lpp promoter-based plasmids in Ty21a even after 50 generations of growth in the absence of antibiotic selection (Table 1), a key quality for vaccine manufacture.

The AVA BioThrax vaccine contains approximately 20 µg of PA per dose (21). Semiquantitative analysis of PA expression in the Salmonella host showed that the amount of PA produced from an optimized PA expression plasmid system, such as the HtrA-PAop-hlyAs system, was approximately 3 µg of PA per 1 x 109 cells. The current Ty21a human dose in the United States can be as high as 6 x 109 CFU, so a comparable human dose for Ty21a producing PA might be roughly equivalent to a dose of the licensed AVA BioThrax vaccine. Moreover, the use of strong in vivo-inducible promoters may result in the production of even higher levels of PA over time in the host. Western blot analysis of PA expression showed that PA was efficiently targeted to the extracellular space by use of a secretion signal, as almost all PA was detected in the supernatant fraction (Fig. 1B). In contrast, PA lacking the HlyA secretion signal was detected mainly in the pellet fraction.

S. enterica serovar Typhi is a human-specific pathogen, and there are no animal models in which to evaluate orally administered, live, attenuated S. Typhi vaccine candidates. However, i.p. immunization and challenge of mice has been employed as a preclinical model to demonstrate immune stimulation by S. Typhi-vectored vaccine candidates. Thus, the immunogenicity and protective efficacy of the Salmonella constructs were evaluated using the A/J mouse model, because anti-PA-based protective efficacy can be demonstrated with this strain (43). In addition, as reported by one of us previously, the anthrax disease progression in this mouse strain challenged with aerosolized Sterne spores is similar to that seen for other species, such as rabbits and nonhuman primates, challenged with fully virulent B. anthracis (23).

Mice were immunized three times, 2 weeks apart, with Ty21a expressing PA under control of the nirB or htrA promoter. These vaccine constructs were able to induce high antibody titers (GMT, 30,000) in A/J mice (Fig. 2). Other recent studies using live attenuated Salmonella expressing PA have not generated such robust anti-PA responses (7, 38) unless the vaccine construct was introduced via the intravenous route (8). Secretion of PA from the host bacterial cell enhances PA-specific serum IgG antibody production, as the vaccine constructs expressing nonsecreted PA induced a GMT of only 600. The lower titers elicited by the construct expressing PA under control of the nirB promoter are due to yields of PA slightly lower than those obtained with the htrA promoter (data not shown).

Several studies using PA-based vaccines in rabbits, guinea pigs, and rhesus macaques have shown that there is not a positive correlation between the amount of total circulating PA-specific IgG and protection against B. anthracis infection. However, a direct correlation was found between the titers of LeTx-neutralizing antibodies and protection against challenge in rabbits (30, 33, 42). High titers (16,241) of toxin-neutralizing antibodies were induced by Ty21a-vectored derivatives secreting PA under control of the htrA promoter and administered i.p. (Table 2). In our model system, i.n. administration of similar vaccine candidates elicited significantly lower toxin-neutralizing titers (2,412) than i.p. administration. Interestingly, use of the Ty21a derivative producing PA intracellularly (HtrA-PAop) resulted in very low titers of toxin-neutralizing antibodies. These observations demonstrate the importance of PA secretion by the bacterial vector, suggesting that the immune system cells can more easily access and process the secreted antigen. We appreciate the fact that the modified PA protein (without a furin cleavage site and with a noncleaved secretion signal at the C terminus) might affect the overall structure of PA and could result in elimination of important neutralizing epitopes. However, our constructs stimulated high serum anti-PA antibody titers (Fig. 2) and high LeTx-neutralizing titers (Table 2) in vaccinated mice.

Protective immunity induced by the Salmonella-vectored PA vaccines was evaluated by i.n. challenging immunized mice with a lethal dose (>20 LD50) of aerosolized spores of nonencapsulated, toxin-producing B. anthracis Sterne strain 7702. Two different vaccine constructs producing secreted PA were tested in this study, and they generated a wide range of toxin-neutralizing antibody titers (1,702 to 16,241). Despite the wide range of toxin-neutralizing titers, all vaccine constructs producing secreted PA (regardless of i.n. or i.p. administration) provided complete protection against a lethal spore challenge. In contrast, only 40% of mice immunized with Ty21a producing PA intracellularly were protected, and this is a reflection of the low titer of functional antibodies produced in these animals (Table 2). As expected, control mice immunized with the Ty21a vector alone were not protected, and all of them died within 5 days following challenge, a profile identical to that of naïve mice challenged in the same manner (23). Two independent mouse immunogenicity (Fig. 2) and challenge (Fig. 4) studies were performed, and they yielded virtually identical results. LeTx-neutralizing titers, as shown in Fig. 3 and Table 2, were determined only in a single mouse immunization study.

The data demonstrate that S. enterica serovar Typhi strain Ty21a can serve as a suitable host for efficient production, secretion, and delivery of anthrax PA. Use of Ty21a exploits the extensive safety record of this existing licensed live attenuated oral typhoid vaccine, which has been administered to over 200 million individuals over 25 years with no documented cases of reversion to virulence or the occurrence of significant adverse reactions, such as reactive arthritis. In addition, field trials of Ty21a in developing countries have demonstrated generation of immunity lasting more than 7 years with 70 to 80% efficacy, depending on the formulation (19). Recent collaborative studies with Aridis Pharmaceuticals LLC (San Jose, CA) have resulted in the manufacture of temperature-stabilized dried preparations of Ty21a that have a projected shelf life of 5 to 10 years at 4°C and ≥1 year at room temperature. Thus, this vaccine has potential for inexpensive production, a long shelf life, and distribution without refrigeration, valuable qualities for a biodefense vaccine.

The mouse has served as a suitable preliminary model for evaluating the immunogenicity and protective efficacy of the vaccine constructs. Two previous studies using attenuated Salmonella to deliver PA in mice have relied on S. enterica serovar Typhimurium as a delivery platform for PA (8, 38). However, a Salmonella-based oral vaccine against anthrax infection in humans will most likely depend on Salmonella serovar Typhi as a delivery vector. One previous study showing expression of PA in S. Typhi reported only moderate immune responses against PA in vaccinated mice and provided no data on protective efficacy in this animal model (7). Here we show that the safe, licensed typhoid vaccine strain S. Typhi Ty21a can efficiently deliver optimized anthrax PA to induce a robust protective immune response against a lethal aerosolized anthrax spore challenge in vaccinated mice. The ultimate goal of these studies, a temperature-stable, oral human vaccine against anthrax infection that can be self-administered in a few doses over a 1-week period, now awaits further animal (rabbits) and phase I human testing.


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ACKNOWLEDGMENTS
 
We thank Steve H. Leppla (National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD) for providing materials and advice. We also thank Jay Slater and Karen Meysick for critical reading of the manuscript.

This work was supported by a grant from the Trans-NIH/FDA Biodefense Program.

The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any agency determination or policy.


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FOOTNOTES
 
* Corresponding author. Mailing address: 29 Lincoln Dr., Building 29, Room 431, Bethesda, MD 20892. Phone: (301) 435-4458. Fax: (301) 480-5527. E-mail: manuel.osorio{at}fda.hhs.gov Back

{triangledown} Published ahead of print on 29 January 2009. Back

{dagger} Supplemental material for this article may be found at http://iai.asm.org/. Back

Editor: S. R. Blanke

{ddagger} Present address: National Institutes of Child Health and Human Development, NIH, Bethesda, MD. Back


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Infection and Immunity, April 2009, p. 1475-1482, Vol. 77, No. 4
0019-9567/09/$08.00+0     doi:10.1128/IAI.00828-08
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