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Infection and Immunity, July 2005, p. 4043-4053, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4043-4053.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Oral Spore Vaccine Based on Live Attenuated Nontoxinogenic Bacillus anthracis Expressing Recombinant Mutant Protective Antigen

R. Aloni-Grinstein,1 O. Gat,1 Z. Altboum,2 B. Velan,1 S. Cohen,1 and A. Shafferman1*

Department of Biochemistry and Molecular Genetics,1 Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona 74100, Israel2

Received 13 January 2005/ Returned for modification 15 February 2005/ Accepted 27 February 2005


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ABSTRACT
 
An attenuated nontoxinogenic nonencapsulated Bacillus anthracis spore vaccine expressing high levels of recombinant mutant protective antigen (PA), which upon subcutaneous immunization provided protection against a lethal B. anthracis challenge, was found to have the potential to serve also as an oral vaccine. Guinea pigs immunized per os with the recombinant spore vaccine were primed to B. anthracis vegetative antigens as well as to PA, yet only a fraction of the animals (30% to 50%) mounted a humoral response to all of these antigens. Protective immunity provided by per os immunization correlated with a threshold level of PA neutralizing antibody titers and was long-lasting. Protection conferred by per os immunization was attained when the vaccine was administered in the sporogenic form, which, unlike the vegetative cells, survived passage through the gastrointestinal tract. A comparison of immunization of unirradiated spores with immunization of {gamma}-irradiated spores demonstrated that germination and de novo synthesis of PA were prerequisites for mounting an immune protective response. Oral immunization of guinea pigs with attenuated B. anthracis spores resulted in a characteristic anti-PA immunoglobulin isotype profile (immunoglobulin [G1 IgG1] versus IgG2), as well as induction of specific anti-PA secretory IgA, indicating development of mucosal immunity.


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INTRODUCTION
 
Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. Three disease forms are recognized in humans, depending on the route by which spores enter the body; these three forms are the cutaneous form (via skin infection), the pulmonary form (via airborne inhalation), and the gastrointestinal form (via ingestion of contaminated food) (29). Fatal inhalational anthrax was a major concern during the recent deliberate B. anthracis spore dispersal events (38), which emphasized the need to focus on providing local immune protection at the mucosal sites of invasion in addition to systemic protection.

The major B. anthracis virulence factors are encoded by two plasmids, pXO2, which carries the genes directing the synthesis of the poly-D-glutamic acid capsule, and pXO1, which encodes the two binary exotoxins, the lethal toxin (LT) and the edema toxin (51, 52). The two toxins have a common cell receptor-binding component, the protective antigen (PA), which interacts with the lethal factor (LF) and the edema factor (EF) to form LT and edema toxin, respectively. After binding to the cell receptor, PA mediates the translocation of LF and EF into the cytosol, where they have their detrimental activities. PA has an essential role in the induction of immunity and protection against the disease, and vaccination with PA alone can induce protective immunity (2, 21, 65). There is a direct relationship between the amount of PA administered to experimental animals and the extent of the humoral immune response elicited against PA (11, 39, 40, 43, 58, 65). PA neutralizing antibody titers, measured by in vitro protection of macrophage cell lines from toxicity by LT, were shown to correlate with the in vivo protective immunity (58).

Two PA-based acellular vaccine formulations have been licensed for human use, one in the United States and one in the United Kingdom. Both consist mainly of PA from cultures of nonencapsulated, toxin-producing B. anthracis strains, and they are adsorbed onto aluminum hydroxide gel and alum precipitated, respectively (32, 46). The United States vaccine is administered subcutaneously (s.c.) (13), and the United Kingdom vaccine is given intramuscularly (anthrax vaccing PL1511/0037, product reference no. D. 1031 [1979]; prepared for the Department of Health and Social Security, London, United Kingdom, by the Public Health Laboratory Service/Centre for Applied Microbiology and Research, Porton Down, Salisbury, United Kingdom). These vaccines provide significant systemic protection against anthrax infection (17, 32) but require multiple doses and annual immunization to maintain immunity (8). This underscores the need for an improved vaccine that induces immunity rapidly and provides longevity with less frequent immunization, using a convenient route of administration.

Three major approaches have been used to generate an improved efficacious anthrax vaccine. The first approach was improvement of the current anthrax acellular PA vaccines by examining various adjuvants (31, 32, 35, 37, 50). The second approach was inclusion of additional bacterium-derived components either by conjugating the poly({gamma}-D-glutamic acid) component of the capsule to recombinant PA (59, 63) or by adding inactivated spores (9). Finally, the third approach was to use live attenuated B. anthracis strains (3, 11, 23, 34, 36, 49, 55, 56). Indeed, experiments performed in our laboratory established that live attenuated B. anthracis vaccine strains, expressing high levels of recombinant native or mutant PA versions (designated MASC-10 and MASC-12/13, respectively [11, 49]), provide effective protective immunity against anthrax in a guinea pig model for at least 12 months following a single subcutaneous immunization. The long-lasting immunity is probably the result of the fate of attenuated vaccine spores in the vaccinated animals, which allows prolonged presentation of low doses of antigens to the immune system (11, 49). The use of a live attenuated bacterial vaccine offers many potential advantages, such as bacterium-enhanced recombinant PA presentation to the immune effector cells and exposure of the immune system to additional potentially protective spore and vegetative bacterial antigens.

Immunization via mucosal routes is thought to be more appropriate for combating diseases caused by pathogens that invade through mucosal surfaces (6, 47). Several studies have reported nasal immunization of mice with PA formulated with a potent mucosal adjuvant (7, 24) or associated with microspheres (19). Others workers have explored the potential of the oral route using B. anthracis Sterne spores (57) or various strains of Salmonella (12, 22) or Lactobacillus casei (68) as expression and delivery systems for PA.

The present study was undertaken to evaluate the protective efficacy of the B. anthracis MASC-13 live attenuated vaccine when it is administered via the oral route. The data presented here provide evidence that the efficacious live attenuated MASC-13 spores may serve as an oral vaccine that is able to elicit a protective immune response when it is administered per os.


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MATERIALS AND METHODS
 
Preparation of B. anthracis live attenuated vaccine. MASC-13 vaccine (49) is based on the attenuated B. anthracis {Delta}14185 strain (11), which constitutively expresses, via the {alpha}-amylase promoter, high levels of an inactivated form of recombinant PA (mrPA313) with deletions of residues F313 and F314 of wild-type PA. Such mutations were reported to result in an inability to translocate LF and EF into the cytosol (41, 64). MASC-13 spores were produced in SSM broth as described previously (11), except that the spores were washed four times with sterile water, suspended in water, and kept at –70°C until they were used.

MASC-13 vegetative cells were grown in modified FA medium (3.3% tryptone, 2% yeast extract, 0.74% NaCl, 0.4% KH2PO4, 0.8% Na2HPO4, 2% glycerol; pH 8) supplemented with 25 µg/ml kanamycin to an A550 of 6, harvested, suspended in phosphate-buffered saline (PBS) to a concentration of 5 x 108 CFU/ml, and used immediately for vaccination.

PA acellular vaccine. The PA vaccine was prepared by adsorption of purified PA (final concentration, 50 µg/ml) to an aluminum hydroxide gel as described previously (58).

Guinea pigs. Female Hartley guinea pigs (weight, 220 to 250 g) were obtained from Charles River Laboratories (United Kingdom). Studies were performed in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals (1997) and were approved by the Animal Use Committee of the Israel Institute for Biological Research.

Immunization and challenge. Guinea pigs were immunized either orally or subcutaneously with various doses of spores or vegetative cells. Oral immunizations were carried out by a noninvasive drop-feeding procedure; the liquid bacterial preparations (1 ml) were gently pipetted into the mouths of the animals, which were allowed to swallow the entire volume. For each oral immunization, spores were divided into three sequential doses given on days 0, 2, and 4 (unless indicated otherwise). Sham vaccination was performed by giving animals 1 ml water. Serum samples were collected from all animals at different times. Animals were challenged subcutaneously with 20 to 40 50% lethal doses (LD50) of B. anthracis strain Vollum (1 LD50 was 100 spores). Prior to challenge the spores were heat shocked for 20 min at 70°C. Animals were observed for 3 weeks after the challenge.

Analysis of viable bacteria in fecal samples. Feces of animals were sampled on days 1, 2, 5, and 13 after oral immunization. For each time, droppings were collected for 24 h in cages containing six animals each. The average amount of bacteria secreted per animal per day was then calculated. The feces were suspended in 10 volumes of PBS, and bacterial counts were determined by plating serial dilutions on Luria-Bertani (LB) agar (Difco) plates containing kanamycin (25 µg/ml). These counts included both vegetative cells and spores. To determine the numbers of spores in the fecal samples, the same samples were heat shocked for 20 min at 70°C prior to plating.

Survival of MASC-13 spores and vegetative cells in gastric fluid. MASC-13 vegetative cells were grown in modified FA medium as described above, harvested, and suspended in PBS to obtain 5 x 108 CFU/ml. Vegetative cells or spores (5 x 108 CFU) were suspended in either 1 ml of PBS or 1 ml of freshly drawn gastric fluid from guinea pigs. The suspensions were incubated at 37°C, removed at different times, serially diluted, and plated on LB agar containing kanamycin (25 µg/ml) to determine viable counts (CFU/ml).

{gamma} Irradiation of spores. Spores were gamma irradiated with a cobalt 60 irradiator (type JS-6500; Nordion, Canada) at the Sor-Van radiation center (Kiriat Soreq, Yavne, Israel). A linear dose-response curve for survival was determined, and a total dose of 2 megarads (0.1 megarad per h) was selected for reduction of viable counts from 1.5 x 109 CFU/ml to 350 CFU/ml.

Preparation of antigens for immunological tests. For enzyme-linked immunosorbent assays (ELISA), the following vegetative antigen preparations were used. (i) Recombinant PA was produced and purified as previously described (11).

(ii) A membrane-enriched fraction of B. anthracis {Delta}14185, designated Mb1, was prepared from a culture grown in Difco brain heart infusion medium (Becton Dickinson) at 37°C to an A550 of 20 to 24. A wet bacterial pellet was suspended in 40 mM Tris buffer (pH 8) supplemented with protease inhibitors (Sigma, St. Louis, Mo.) and briefly sonicated. Following three washes of the pellet with 40 mM Tris buffer (pH 8), the insoluble material was resuspended in 8 M urea in the same buffer and then centrifuged. The supernatant containing 7 mg/ml protein consisted of approximately 50% EA1 protein, as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

(iii) A secreted protein fraction of B. anthracis {Delta}14185 was collected from the bacterial culture described above. The conditioned growth medium was subjected to 10% trichloroacetic acid precipitation (4°C overnight), followed by centrifugation (Sorvall SS34 rotor; relative centrifugal force, 48,000), and the pellet was resuspended at a final concentration of 2 mg/ml protein in 40 mM Tris buffer (pH 8).

For the cell proliferation assays we used purified PA (described above) and an EA1-enriched (80%) membrane fraction which was obtained by subjecting membrane fraction Mb1 (described above) to an additional extraction step with 5 M urea and 2 M thiourea. The final extract, designated Mb2, contained 8 mg/ml protein.

Extraction of antibodies from fecal samples. Fresh fecal pellets were suspended 1:10 in PBS and centrifuged for 20 min at 5,000 x g at room temperature. The supernatant was filtered through a sterile acrodisc low-protein-binding 0.45-µm filter and frozen until it was used.

Detection of specific IgG antibodies. An ELISA for detection of immunoglobulin G (IgG) antibodies against PA and against bacterium-related antigens (membrane and secreted protein fractions) was carried out by adding serial twofold dilutions of sera from immunized guinea pigs to a 96-well microtiter plate (Nunc, Roskilde, Denmark) coated with the relevant antigen. The plates were coated with 350 ng protein/well of recombinant PA and 200 ng protein/well of the Mb1 or secreted fractions in NaHCO3 buffer (50 mM, pH 9.6). Rabbit anti-guinea pig immunoglobulin G conjugated to alkaline phosphatase (Sigma) served as the second antibody. Detection was performed by monitoring p-nitrophenyl phosphate (Sigma) hydrolysis. End point titers were defined as the highest serum dilutions that resulted in a twofold increase compared with the background value.

Detection of IgG subtypes (IgG1 and IgG2). Plates were coated with PA as described above, and serial twofold dilutions of sera from immunized guinea pigs were added. Detection was performed with goat anti-guinea pig IgG1 and goat anti-guinea pig IgG2 conjugated to alkaline phosphatase (Bethyl) at a 1:500 dilution.

Evaluation of anti-guinea pig IgA antibody preparations. Several commercial preparations of antibody against guinea pig IgA alpha chain were examined. These preparations included rabbit antibodies (Bethyl, ICN, and Bionet) and sheep antibodies (Bethyl). Anti-rabbit IgG conjugated to alkaline phosphatase (Sigma) and donkey anti-sheep IgG conjugated to alkaline phosphatase (Jackson Immunoresearch) were used as second antibodies. Antibody specificity was evaluated by comparing reactivity to guinea pig IgA and IgG (Accurate Scientific Corp.) coated onto 96-well plates. Of the four anti-alpha chain preparations tested, we selected the rabbit anti-guinea pig IgA alpha chain from Bethyl, since at a dilution of 1:2,000 it exhibited the highest selectivity (the IgA/IgG signal ratio was 10:1).

Detection of anti-PA IgA antibodies. Serially diluted fecal extracts or sera were applied to PA-coated 96-well microtiter plates. Following 1 h of incubation at 37°C, specific anti-PA IgA antibodies were detected as described above. To minimize nonspecific background readings, both blocking and dilution were performed with 1% gelatin (Merck) rather than with the more commonly used bovine serum albumin-based buffer.

Neutralizing antibodies. Neutralizing antibody titers were determined essentially as described previously (11, 58) by using the abilities of the antibodies to prevent PA-LF-induced mortality of murine macrophage J774A.1 cells (American Type Culture Collection). Aliquots (0.1 ml) of a cell suspension (8 x 105 to 106 cells/ml) were plated onto 96-well culture plates (Nunc). The sera tested were serially diluted in twofold steps in Dulbecco modified Eagle medium supplemented with 5% fetal calf serum containing PA (5 µg/ml) and LF (2 µg/ml). Following 1 h of incubation, 10-µl portions of the serum-toxin complex mixtures were added to the J774A.1 cells. The plates were incubated for 3 h at 37°C in 5% CO2, and cell viability was monitored by a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide-thiazolyl blue assay (the absorbance was measured at 540 nm) (53). End point titers were defined as the highest serum dilutions that resulted in a twofold increase compared with the background value. Neutralizing antibody titers were expressed as reciprocal end point dilutions.

Cell proliferation assays. Blood from naïve and immunized guinea pigs was collected in a CPT Vacutainer with sodium heparin cell preparation tubes (Becton Dickinson, Plymouth, United Kingdom). Peripheral blood mononuclear cells (PBMC) were separated by centrifugation at a relative centrifugal force of 1,500 for 12 min, washed three times with sterile culture medium (RPMI 1640, 10% fetal calf serum, L-glutamine). Cells were seeded into 96-well round-bottom plates (Nunc) at a concentration of 1.5 x 105 cells/well in 0.1 ml culture medium. Recombinant PA (0.12 µg) or Mb2 (the EA1-enriched membrane fraction [0.3 µg]) was then added to cells. The plates were incubated in a humid environment in the presence of 5% CO2 at 37°C for 6 days. The assessment of cell proliferation was based on measurement of bromodeoxyuridine incorporation during DNA synthesis into proliferating cells using the Biotrak cell proliferation ELISA system (Amersham Pharmacia Biotech). A stimulation index was calculated (means of four replicates) by dividing the optical densities measured in a stimulated culture and a nonstimulated culture.


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RESULTS
 
Fate of spores and vegetative cells of the live attenuated recombinant B. anthracis MASC-13 vaccine in orally immunized guinea pigs. The studies reported here were performed with the MASC-13 B. anthracis recombinant strain (49). This is a nontoxinogenic (pXO1–) noncapsulated (pXO2–) B. anthracis {Delta}14185 strain (11) expressing constitutively, by means of the heterologous {alpha}-amylase promoter, high levels (100 µg/ml in vitro) of an inactivated form of recombinant PA (mrPA313) with deletions of residues F313 and F314 of wild-type PA. Such mutations were reported to result in an inability to translocate LF and EF into the cytosol (64). A single immunization of guinea pigs with 5 x 107 spores of this highly attenuated strain was shown recently to provide protective immunity against a lethal challenge even 12 months after subcutaneous immunization (49). It is worth noting that the attenuation level of the recombinant B. anthracis vaccines expressing either PA or a mutant PA derivative is greater than that of the Sterne vaccine (the LD50 of strain Sterne in guinea pigs is 7.5 x 106 spores, while the LD50 of strain MASC-10 or MASC-13 is greater than 5 x 108 spores [10, 11, 49]).

Using the MASC-13 strain, we initiated immunization studies via the oral route. A noninvasive procedure based on drop feeding (see Materials and Methods) rather than intraesophageal needle feeding was employed in order to avoid any possible tissue damage which could result in alternative routes of entry and immunization. This was essential in view of the high efficacy of s.c. immunization and the fact that 102- to 103-fold-higher doses are used for oral immunization.

In a previous study we showed that protective immunity can be achieved by s.c. immunization with either spores or the vegetative form of attenuated B. anthracis strains (11, 49). Thus, as a first step, we examined both spores and vegetative cells of MASC-13 for their potential as orally delivered vaccines. Two groups of six guinea pigs were given single doses equivalent to 5 x 109 vegetative cells or spores of the live attenuated recombinant strain. Pooled fecal samples from each vaccinated group were collected and analyzed for viable secreted B. anthracis spores and cells on LB agar-kanamycin plates at various times. Animals immunized with 5 x 109 spores shed 5 x 108 bacteria in the spore form on the first day postimmunization, which was followed by ongoing but declining secretion for 13 days. In contrast, for animals immunized with vegetative cells, bacteria were barely found in the feces (~2,000 output cells after an input of 5 x 108 cells) on the first day postimmunization (Fig. 1). To determine whether the decrease in the count was due to the susceptibility of the vegetative cells to conditions in the gastrointestinal tract (GIT), we compared the survival of spores and the survival of vegetative cells in gastric fluid. Approximately 5 x 108 CFU/ml MASC-13 vegetative cells or spores were incubated at 37°C in gastric fluid drawn from the guinea pig stomach. Survival was determined by counting the viable bacterial cells (CFU/ml) at various times (Fig. 1, inset). Spores were found to be essentially unaffected by the simulated gastric conditions, whereas a dramatic reduction in the viable counts of vegetative cells was observed within 15 min after the start of incubation (Fig. 1, inset). These results, together with the fecal clearance results (Fig. 1), led us to use the spore form for evaluating the potential of the MASC-13 strain as an oral vaccine.



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  		FIG. 1. Fate of spores and vegetative cells of the MASC-13 vaccine in gastric fluid and in the GIT of guinea pigs. Two groups of six guinea pigs were orally immunized either with 5 x 109 spores (•) or 5 x 108 vegetative cells ({circ}) per animal. A pooled stool sample was analyzed for viable counts at various times. The dotted lines indicate the recorded decline from the initial input to the first point examined. (Inset) Survival of incubated bacteria in gastric fluid at 37°C. Spores (•) or vegetative cells ({circ}) were suspended in gastric fluid drawn from a guinea pig and assayed for viability at different times.

Humoral response and efficacy of the live attenuated recombinant MASC-13 spore vaccine administered by the oral route. Guinea pigs were orally immunized with a total of 1.5 x 1010 MASC-13 spores per animal by three consecutive feedings of 5 x 109 spores on days 0, 2, and 4. A parallel group of guinea pigs was immunized with a single dose (5 x 106 spores) of the same vaccine spore strain via the s.c. route. Blood was withdrawn 6 and 12 weeks postimmunization, and antibody titers against PA were determined (Table 1). The results were the first indication that oral immunization with the live attenuated B. anthracis vaccine can produce a specific humoral response. Of the seven positive responders after per os immunization, four exhibited significant neutralizing antibody titers. The efficacy of the oral vaccination was determined by challenging the animals 13 weeks postimmunization with 20 LD50 of Vollum spores. Only the four animals that had measurable neutralizing antibody titers (≥400) survived the challenge. A similar correlation between neutralizing antibody titers and protection was observed for s.c. immunization (Table 1). However, we noted that s.c. immunization with of 5 x 107 or more attenuated spores could lead to very effective seroconversion and protection of all animals (11, 49), whereas immunization with 1.5 x 1010 spores per os was partially protective.


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  		TABLE 1. Anti-PA antibody response and survival after lethal challenge following a single subcutaneous or per os immunization of guinea pigs with MASC-13 spores

In an attempt to increase the number of immunoresponsive animals treated by the oral route, additional immunization protocols were examined. In the experiment whose results are shown in Table 2, guinea pigs were immunized as described above with a total dose of 1.5 x 1010 spores, and 7 weeks after the primary immunization the guinea pigs were vaccinated again with a single dose consisting of 5 x 109 spores. The immunological responses to various B. anthracis antigens were monitored.


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  		TABLE 2. Antibody titers of individual animals and survival after a lethal challenge following two per os vaccinations

The second immunization appeared to result in an increase in the number of animals exhibiting a humoral response to the membrane antigen fraction (Table 2). However, the number of animals (6 of 12 animals) producing detectable anti-PA antibody titers did not increase following the second immunization, although the level of the neutralizing antibodies appeared to increase to some extent. Of the six animals with measurable neutralizing antibodies, five survived a lethal challenge with Vollum spores. All surviving animals had neutralizing antibody titers greater than 200, a level consistent with the previously determined level of anti-PA neutralizing antibodies required for protective immunity (58). In the following experiment, we examined the effect of a series of four consecutive oral immunizations at weeks 0, 11, 22, and 33 using a total of 1.5 x 1010 spores for every immunization (Table 3). Sera were examined 10 days after each immunization for antibodies against PA, as well as for antibodies against the membrane Mb1 and secreted fractions of B. anthracis {Delta}14185 (we note that this strain does not express PA). Six weeks after primary immunization, about 20% of the animals (Table 3, animals 1 to 3) developed antibodies to all antigens tested. Following a second immunization, no change in the number of animals carrying anti-PA antibodies was noted (as found also in the experiment whose results are summarized in Table 2), but the number of animals producing antibodies to the membrane Mb1 fraction increased, as did the number of animals exhibiting antibodies to secreted antigens. Following the third immunization, the percentage of animals exhibiting anti-PA antibodies increased (to 50%), while no further increase in the percentage of animals producing antibodies for other antigens was found (although increases in titers were observed). Following the fourth immunization, no change in the number of seropositive animals was observed, yet in most of the animals that did develop anti-PA antibodies upon the third immunization, a boost response was noted for each of the antigens tested.


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  		TABLE 3. Antibody response following primary oral immunization and three sequential per os immunizations at 11-week intervalsa

It is worth noting that the immunological response to oral vaccination with the attenuated B. anthracis spores was achieved by a spore dose which was equal to or lower than the doses reported to be effective by the same immunization route for other microorganisms. For example, mice were vaccinated nine times with approximately 1010 Bacillus subtilis spores per dose (15, 16, 44) or seven times with 5 x 109 CFU of Lactococcus lactis per dose (61) in order to generate detectable levels of relevant antibodies.

In summary, following four oral immunizations, 50% of the animals developed antibodies against all the B. anthracis antigens tested (Table 3), and an additional 20% developed antibodies against somatic antigens but not against PA, yet about 30% of the animals did not exhibit any measurable humoral response.

Cellular immune response in guinea pigs orally immunized with MASC-13 spores. In all oral immunization experiments described above, we identified a fraction of animals that did not appear to seroconvert to PA or to other B. anthracis antigens. In order to determine whether all vaccinated animals were primed by the vaccine, a lymphocyte proliferation assay was performed with PBMC obtained from immunized guinea pigs. Proliferation of PBMC was measured following stimulation with PA or with EA1-enriched membrane preparation Mb2 (see Materials and Methods). All immunized animals exhibited a clear positive proliferation response compared to naïve animals, yet higher stimulation indices were measured for seropositive animals. The results of representative experiments are shown in Fig. 2.



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  		FIG. 2. Stimulation indices (S.I.) for PBMC from animals orally immunized with spores for PA and Mb2 antigens. The proliferative responses of PBMC (1.5 x 105 cells/well) from orally immunized guinea pigs to PA and Mb2 were assessed by using the bromodeoxyuridine ELISA. The data are means and standard errors of the means (n = 4) for specific absorbance. (A) Animal exhibiting a humoral response to PA and Mb2 (animal 4 [Table 3]). (B) Animal exhibiting no response to B. anthracis antigens (animal 14). (C) Naïve animal. O.D 450, optical density at 450 nm.

These results suggest that all vaccinated animals were exposed to antigens derived from the vegetative form of the bacteria.

De novo expression of PA in guinea pigs is a prerequisite for induction of the protective response by the live spore vaccine. The anti-PA response after MASC-13 vaccination could have been driven by PA produced and secreted from B. anthracis following germination or by preformed PA present in our spore preparation either as a contaminant or as a genuine constituent of the spore wall, as suggested by Welkos et al. (67). To discriminate between these possible alternatives, we compared the immunological responses to {gamma}-irradiated inactivated spores and live unirradiated spores. The s.c. route of immunization was chosen since this mode of immunization elicits an efficient and uniform immune response to B. anthracis antigens at relatively low doses (11).

The irradiation dose selected led to a 107-fold reduction in viability (see Materials and Methods) with no observable damage to the spore morphology, as determined by microscopic examination. Two groups of guinea pigs were immunized with either 5 x 107 spores per animal or the equivalent amount of {gamma}-irradiated spores. Antibody titers to PA, Mb1, and the secreted fractions of the {Delta}14185 strain were determined 6 weeks postimmunization for each animal (Table 4). Animals that were vaccinated with unirradiated spores (which were able to germinate and to form colonies on agar plates) produced very high levels of PA antibodies (geometric mean titer [GMT], 4,800), while animals vaccinated with the {gamma}-irradiated inactivated spores had marginal levels of anti-PA antibodies (GMT, 45) (Fig. 3). In contrast, both {gamma}-irradiated and unirradiated spores resulted in high titers to the membrane or secreted {Delta}14185-derived antigens. As expected, the titers to these antigens were somewhat higher in animals immunized with the unirradiated spores (Fig. 3 and Table 4). These results provide evidence that induction of the anti-PA response is triggered by de novo synthesis of PA in the vaccinated animals and is not the result of carryover of contaminants due to the possible limited PA mass suggested to be endogenous to the spore (67).


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  		TABLE 4. Antibody titers (ELISA) to specific spore vaccine antigens and protection following s.c. immunization with unirradiated and {gamma}-irradiated MASC-13 spores



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  		FIG. 3. Comparison of humoral responses following s.c. immunization with irradiated and unirradiated spores: GMTs to PA and other B. anthracis antigens, membrane fraction Mb1, and secreted fractions (Sec.). Individual data are shown in Table 4. Black bars, unirradiated spores; gray bars, irradiated spores.

Vaccinated animals were challenged 6 weeks postimmunization with a lethal dose of Vollum spores. None of the animals vaccinated with {gamma}-irradiated spores survived the challenge, and none showed any delay in the mean time to death compared to naïve animals. On the other hand, all animals vaccinated with unirradiated spores survived the challenge (Table 4). These results suggest that germination and PA production by the vaccine spores is essential for the development of an efficient immune response that leads to full protection against a lethal anthrax challenge.

Type-specific immunoglobulin response following oral immunization with the live vaccine. The specific anti-PA IgG subclass pattern obtained following oral immunization with the live attenuated MASC-13 vaccine was compared to the pattern obtained following immunization with an acellular PA-based vaccine. While s.c. vaccination with the acellular antigen led to a dominant IgG1 response, in all animals in which seroconversion to anti-PA had occurred (titer range, 1,280 to 10,240) following oral spore immunization a clear anti-PA-specific dominant IgG2 response was observed (Table 5). In this experiment no difference was found between animals exposed to live MASC-13 vaccine by the oral route and animals exposed to live MASC-13 vaccine by the s.c. route. These results may suggest that live vaccine immunization triggers a substantial Th1-type immune response (see Discussion).


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  		TABLE 5. Antibody subtyping following vaccination with MASC-13 spores

The hallmark of mucosal vaccination is the production of secreted antibodies of the IgA type, which is believed to provide mucosal protection (54). Indeed, oral vaccination with MASC-13 spores led to secretion of specific anti-PA IgA antibodies into the GIT lumen, as manifested by the recovery of such antibodies from the feces of vaccinated animals (Fig. 4). The presence of such antibodies in animal sera could not be evaluated due to the extensive cross-reactivity of anti-guinea pig IgA with IgG (see Materials and Methods).



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  		FIG. 4. Induction of IgA-specific response following oral vaccination with MASC-13 spores. The data are for animals having circulatory anti-PA antibody titers of 1,280 to 10,240 (Table 2, animals 1 to 4). (A) Ratio of anti-PA IgA antibodies to anti-PA IgG antibodies in feces at various dilutions of fecal extract. (B) Ratio of anti-PA IgA antibodies to anti-PA IgG antibodies in sera. Note that the anti-PA IgA antibodies are at the limiting level of detection due to the limitation of the commercially available reagents. O.D405, optical density at 405 nm.


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DISCUSSION
 
Problems encountered with the available anthrax vaccine underline the need for an improved vaccine formulation. The optimal vaccine should be effective and easy to administer to large populations and should target protection against the potential anthrax bioterror scenarios, namely, infection through the upper respiratory tract (inhalation) and the gastrointestinal tract (food and water consumption). In an attempt to address these requirements, we developed several nontoxinogenic noncapsular attenuated anthrax strains which express high levels of recombinant native or mutant PA (11, 49). We have shown previously that a single s.c. immunization with spores of such strains (MASC-10 and MASC-13) provide long-lasting immunity in a guinea pig model against a high lethal challenge (11, 49). The potential application of these attenuated strains as vaccines could increase substantially if they are proven to be effective upon oral administration.

Data presented in this paper provide evidence that live attenuated B. anthracis MASC-13 spores may have the potential to serve as a vaccine when they are administered per os. We first showed that the MASC-13 spore, in contrast to the vegetative form, survives the harsh environment of the GIT (Fig. 1). In vitro experiments indicated that MASC-13 spores, but not vegetative cells, survive incubation in fluids withdrawn from the guinea pig stomach. This finding was mirrored in in vivo studies; the mean cumulative counts recovered in the feces of animals 24 h after vaccination with the vegetative forms of B. anthracis were extremely low (0.0004% of the dose). In contrast, ~10% of the initial spore dose was recovered in the feces 24 h after vaccination (Fig. 1). It is interesting that essentially similar observations were made in a different study conducted with a B. subtilis strain used for oral delivery of antigens, in which spores, in contrast to vegetative cells, were highly resistant to simulated gastric fluids and survived in the GIT (14).

The passage of viable spores through the guinea pig GIT results in a rather effective humoral response to various somatic and secreted antigens, as well as to the major protective antigen (Tables 1 to 3). This is accompanied by protective immunity against challenge with a lethal dose of the virulent Vollum B. anthracis strain. As established in other anthrax vaccination studies (33, 39, 58), we found a direct correlation between the presence of anti-PA antibodies in the circulation and survival after a lethal challenge with virulent B. anthracis spores. We observed that the threshold level for full protection is a neutralizing titer of about 200. The same values were observed for s.c. vaccination with an acellular PA-based vaccine (43, 58) and for vaccination with MASC-13 spores administered s.c. (Table 1), attesting to the major contribution of the anti-PA humoral response to protection independent of the mode of presentation of this potent protective antigen.

Another asset of oral vaccination with MASC-13 is the establishment of long-lasting protective immunity. Vaccinated animals reimmunized at week 7 were resistant to challenge 35 weeks after the primary immunization (Table 2). In this respect oral immunization resembles s.c. immunization with live attenuated spores, for which similar longevities were observed (11, 49).

Oral vaccination with attenuated B. anthracis spores appears to differ, however, from s.c. vaccination in the proportion of animals that develop measurable anti-PA antibodies and protective immunity. While s.c. vaccination with the effective spore dose results in protection of all vaccinated animals (11), oral vaccination consistently has a partial effect. Interestingly, increased doses and numbers of vaccination were not effective for establishing full protection (Tables 1, 2, and 3). One could attribute this to genetic variation in the guinea pig population, yet the uniformity in the responses upon s.c. vaccination with the same spore formulation argues against this. Moreover, the partial response does not reflect some inherent genetic deficiency in the immunological response to orogastric antigen presentation, since most of the animals developed specific antibodies to other B. anthracis antigens (Tables 2 and 3). In this context, we noted that while circulatory antibodies to PA were detected only in some of the vaccinated animals, in all animals tested high stimulation indices for PBMC exposed to PA were observed regardless of the presence of detectable antibodies (Fig. 2). This again indicates that in all orally immunized animals presentation of B. anthracis antigens, including PA, did occur.

The apparent difference between the antibody response to PA and the antibody response to somatic anthrax antigens raises the issue of the mechanisms of mounting an immune response upon spore vaccination. To address this, we compared vaccination with live attenuated B. anthracis spores and vaccination with a spore preparation subjected to inactivating {gamma}-irradiation (Table 4 and Fig. 3). While both preparations induced an effective antibody response to the somatic and secreted antigens, live spores were needed to induce anti-PA antibodies. Thus, it appears that while induction of a response to the somatic antigen does not entail de novo synthesis and that a response could be induced directly by antigens present in the vaccine spore formulation, presentation of PA requires germination of the spore and production of a toxin component by a viable vegetative cell. Germination of spores could occur in the GIT lumen (27, 45); however, under these conditions PA production may not be very efficient since vegetative cells are expected to be eliminated, as suggested by the fact that no vegetative forms were recovered in the feces. An alternative explanation involves uptake of the viable spores by professional phagocytic cells (54) lining the intestines, followed by germination, PA production, and then presentation of the de novo-produced antigens. The existence of such a pathway is supported by the well-documented observation that B. subtilis spores could germinate efficiently in macrophages and initiate gene expression (16). The primary events of such a presentation process appear to be rather effective, as judged by the fact that all orally vaccinated animals were found to be primed to PA. Yet the translation of the primary event into a broad antibody response appears to be somehow limited, as judged by the partial humoral response in the orally vaccinated animals. Partial immune response effects were observed in other studies in which antigens were presented orally to various animal models (22, 26, 42, 46, 60, 66). This could be a reflection of the gut-associated "tolerance" (62) that is believed to down-regulate responsiveness to bacterial populations in the intestinal tract, which could be more pronounced in guinea pigs.

Various approaches or a combination of approaches can be examined to overcome the observed partial humoral response with the MASC-13 oral spore vaccine; these approaches include (i) generation of a strain expressing higher levels of PA through utilization of promoters that are much more potent than {alpha}-amylase (23), (ii) coadministration with mucosal adjuvants (5, 25), (iii) engineering the spores with M cells targeting molecules that potentially improve the uptake of the spores by M cells (4, 28), and (iv) development of strains which are able to persist longer in phagocytic cells (18).

The oral vaccination regimen appears to activate multiple arms of the anti-PA immune response. Dissection of the response to PA revealed the presence of circulatory antibodies, as well as cellular memory manifested by generation of cells responsive to in vitro stimulation by PA. In this respect the response to oral vaccination by attenuated B. anthracis spores resembles the response observed upon injection of acellular PA preparations (43, 58), as well as injection of attenuated spores (11, 49; unpublished data). Nevertheless, a careful examination of the nature of the humoral response to oral spore vaccinations revealed some unique features. In contrast to vaccination with acellular PA vaccine, in which a dominant IgG1 response was noted, live vaccine spores induce a dominant IgG2 response when they are administered both orally and s.c. (Table 5). A switch in the IgG subtype is well characterized in mice and is implicated in a switch from a Th1 response to a Th2 response. It is not known whether guinea pigs have a Th1-Th2 system similar to the system described in mice. Nevertheless, it is interesting that when guinea pigs and mice were immunized in parallel with various acellular PA formulations, a switch from IgG1/IgG2a in mice was mirrored by a switch from IgG1/IgG2 in guinea pigs (20, 46). It is therefore tempting to speculate that immunization with MASC-13 may trigger a substantial Th1-type immune response.

The other significant feature of the response to oral spore vaccination is the development of PA-specific IgA antibodies detectable in feces, suggesting that they are secreted into the GIT lumen. Thus, oral vaccination with MASC-13 exhibits the hallmark of mucosal vaccination, namely, production of antibodies at the presumed site of pathogen entry, serving as the first line of defense. As discussed above, this is complemented by an effective systemic response manifested by circulatory IgG and memory cells, both of which serve as the second line of defense against bacteria which succeed in transiting the mucosal barrier.

Numerous attenuated microorganisms have been suggested as oral vaccines per se or as delivery systems for antigen presentation through the GIT. These microorganisms include Salmonella spp., Shigella spp., Yersinia enterocolitica, Listeria monocytogenes, Lactobacillus spp., and Vibrio cholerae, as well as the toxinogenic B. anthracis Sterne strain (48). More recently, spores of B. subtilis were evaluated as presenters of antigens displayed on their surface (14-16, 30, 44).

The B. anthracis spore vaccine developed by us is highly attenuated, yet it is still able to serve as an effective live vaccine when it is administered s.c (11, 49), as well as when it is administered per os. We therefore suggest utilization of this attenuated B. anthracis spore vaccine as an orogastric presentation system for both homologous (11, 49) and heterologous antigens (23). Such antigens may be either surface-exposed antigens or antigens expressed de novo by the bacterium upon invasion (Fig. 3 and Table 4). The proposed oral vehicle could be advantageous since it is derived from a pathogen believed to invade via the orogastric route (and thus is able to present antigens after it penetrates the GIT lining) and at the same time exhibits the resistance of a spore-forming microorganism. The use of such vaccines in oral vaccination is expected to raise a local secretory humoral response, as well as a systemic response. This approach could be suitable for protection against bioterror agents, where infection through mucosal barriers (inhalation and ingestion per os) is probable.


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ACKNOWLEDGMENTS
 
We thank Y. Sholmovich, G. Friedman, I. Inbar, and N. Seliger for their excellent technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: P.O. Box 19, 74100 Ness-Ziona, Israel. Phone: 972-8-9381595. Fax: 972-8-9401404. E-mail: avigdor{at}iibr.gov.il. Back

Editor: D. L. Burns


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