Immunization against anthrax using Bacillus subtilis spores expressing the anthrax protective antigen.

Protective immunity to anthrax can be achieved by antibodies raised against the secreted protective antigen (PA) and this forms the basis of the current acellular vaccines for human use. Bacillus subtilis spores have previously been used for delivery of heterologous antigens by the oral and nasal routes and their intrinsic heat-stability make them attractive vaccine vehicles. In this study we have expressed PA, or segments of PA, in B. subtilis using two strategies. First, display on the spore coat, and second, in the germinated spore (or vegetative cell). Using parenteral delivery we show that recombinant spores can be used to confer protective immunity in a murine model using an in vitro toxin neutralization assay and a challenge experiment with the latter showing protection to 100 median lethal dose of B. anthracis spores. PA must be secreted from the live bacterium or alternatively displayed on the spore surface to confer protective immunity. Intracellular expression of PA failed to confer protective immunity. The highest levels of protective immunity were achieved when PA was displayed on the spore surface as well as in the germinating spore.

The intestinal life cycle of Bacillus subtilis and close relatives.

Bacillus subtilis is considered a soil organism for which endospore formation provides a means to ensure long-term survival in the environment. We have addressed here the question of what happens to a spore when ingested. Spores displaying on their surface a heterologous antigen, tetanus toxin fragment C (TTFC), were shown to generate anti-TTFC responses not to the antigen contained in the primary oral inoculum but to those displayed on spores that had germinated and then resporulated. We then used reverse transcription-PCR to determine expression of vegetative genes and sporulation-specific genes in the mouse gut following oral dosing with spores. Significant levels of germination and sporulation were documented. Using natural isolates of B. subtilis that could form biofilms, we showed that these strains could persist in the mouse gut for significantly longer than the laboratory strain. Moreover, these isolates could grow and sporulate anaerobically and exhibited a novel phenomenon of being able to form spores in almost half the time required for the laboratory isolate. This suggests that spores are not transient passengers of the gastrointestinal tract but have adapted to carry out their entire life cycle within this environment. This is the first report showing an intestinal life cycle of B. subtilis and suggests that other Bacillus species could also be members of the gut microflora.

Carotenoids present in halotolerant Bacillus spore formers.

Six isolates of pigmented spore-forming bacteria were recovered from human faeces from subjects in Vietnam. 16S rRNA analysis demonstrated close association with known pigmented Bacillus species. All isolates were able to tolerate growth on 8% NaCl and were resistant to arsenate, characteristics that make them most related to Bacillus indicus. Two visible pigments were apparent, a yellow pigment found in vegetative cells and an orange pigment found only in spores. We used high-performance liquid chromatography to characterize and quantify these pigments and found them to be carotenoids. The biosynthetic pathway that generates them branches with one that could lead to the spore-associated orange pigmentation. Although these bacteria were found in faeces, the seafood-rich diet of Vietnam and the recovery of other pigmented Bacillus species from seafood and marine environments makes it highly probable that the true origin of these bacteria is from ingested seafood.

Cases of emesis associated with bacterial contamination of an infant breakfast cereal product.

A commercial product for infants containing cereal mixed with dried infant formula was diagnosed as producing rapid projectile vomiting in two infants. Analysis of multiple samples of the cereal product revealed significant contamination with two spore-forming species, Bacillus subtilis and a strain of Bacillus cereus. The latter is the most likely cause of the emetic food poisoning, but we were unable to detect B. cereus emetic toxin. This raises the possibility of the cause being either a new cereulide-type toxin, or the bacterial load, in which case the presence of B. subtilis could have been a contributing factor.

Intracellular fate and immunogenicity of B. subtilis spores.

To support our work on the development of bacterial spores as oral vaccines we examined the immunogenicity and intracellular fate of Bacillus subtilis endospores in a murine model. Mice dosed orally with spores developed systemic IgG and mucosal sIgA responses. Analysis of IgG subclasses revealed a predominance of the IgG2a subclass during the early stages of immunisation. Analysis of cytokine mRNA in GALT and lymphoid organs showed early induction of IFN-gamma, a Th1 cytokine, as well as the pro-inflammatory cytokine TNF-alpha. Significant levels of IgG antibodies were produced against vegetative bacilli following dosing with spores. This showed that spores could germinate in the GI tract. In vitro studies detailing the intracellular fate and persistence of spores in a macrophage-like cell line (RAW264.7) demonstrated that spores could germinate efficiently in macrophages, initiate gene expression as well as inducing pro-inflammatory cytokines.

Characterization of Bacillus probiotics available for human use.

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.

Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner.

We report the use of CotC, a major component of the Bacillus subtilis spore coat, as a fusion partner for the expression of two heterologous antigens on the spore coat. Recombinant spores expressing tetanus toxin fragment C (TTFC) of Clostridium tetani or the B subunit of the heat-labile toxin of Escherichia coli (LTB) were used for oral dosing and shown to generate specific systemic and mucosal immune responses in a murine model. This report, expanding the previously described expression of TTFC on the spore surface by fusion to CotB [J Bacteriol 183 (2001) 6294] and its use for oral vaccination [Infect Immun 71 (2003) 2810] shows that different antigens can be successfully presented on the spore coat and supports the use of the spore as an efficient vehicle for mucosal immunisation.

Bacterial spores as heat stable vaccine vehicles.

Recently, the first use of bacterial spores as vaccine vehicles was reported, showing that mice orally immunised with Bacillus subtilis spores expressing a tetanus antigen could be protected against lethal challenge with tetanus toxin. Unlike many second generation vaccine systems currently under development, none offer the heat stability of bacterial spores or the flexibility for genetic manipulation. The current use of Bacillus spores as probiotics for both humans and animals may facilitate their eventual licensing as oral vaccines. This review reports the progress that has been made in the establishment of bacterial spores as vaccine vehicles and outlines the potential advantages of the spore vaccine approach.

Germination of the spore in the gastrointestinal tract provides a novel route for heterologous antigen delivery.

We have evaluated the potential of endospores of the Gram-positive bacterium Bacillus subtilis as an oral vaccine delivery system. The key features of the B. subtilis spore as a vaccine are, non-pathogenicity, advanced cloning tools, extreme robustness, long-term storage properties and its current use as a probiotic for both humans and animals. We have shown previously that the spore germinates in the small intestine of the mouse and have exploited this attribute for heterologous antigen delivery in this work. The first part of this study was to evaluate the fate of spores in vivo as well as under simulated gut conditions. This showed that spores were extremely robust with most being excreted in the faeces. Using a recombinant gene expressing high levels of beta-galactosidase specifically in the germinated spore (vegetative cell) we showed that spores administered orally could elicit beta-galactosidase-specific local and systemic immune responses. This demonstrated proof of principle that the germinating spore might be effective in the safe delivery of antigens across the stomach barrier. Interestingly, analysis of IgG subclasses suggested a potential bias towards a Th1 response and the involvement of cellular immunity.

Bacterial spores as vaccine vehicles.

For the first time, bacterial spores have been evaluated as vaccine vehicles. Bacillus subtilis spores displaying the tetanus toxin fragment C (TTFC) antigen were used for oral and intranasal immunization and were shown to generate mucosal and systemic responses in a murine model. TTFC-specific immunoglobulin G titers in serum (determined by enzyme-linked immunosorbent assay) reached significant levels 33 days after oral dosing, while responses against the spore coat proteins were relatively low. Tetanus antitoxin levels were sufficient to protect against an otherwise lethal challenge of tetanus toxin (20 50% lethal doses). The robustness and long-term storage properties of bacterial spores, coupled with simplified genetic manipulation and cost-effective manufacturing, make them particularly attractive vehicles for oral and intranasal vaccination.