Bacterial Spores as Vaccine Vehicles

Abstract

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. This paper reports the first steps in the development of Bacillus subtilis endospores as novel vaccine vehicles. The development of improved vaccination strategies has always been of the utmost importance for a number of reasons: to provide better levels of local immunity against pathogens which enter the body primarily through the mucosal surfaces, to provide needle-less routes of administration, to offer improved safety and minimal adverse side effects, and finally, to provide economical vaccines for developing countries where suboptimal storage and transportation facilities can prevent effective immunization programs. Considerable progress has been made with the development of improved mucosal vaccination strategies as well as novel delivery systems (19). Oral immunization with soluble antigens has long been known to generate poor immune responses due to antigen degradation in the stomach, limited absorption, and tolerance. To improve mucosal immune responses, a number of unique carrier systems have been developed which fall into two general categories, nonliving and living. Nonliving systems include liposomes, microparticles, immunity-stimulating complexes, and formulations based on cholera toxin and Escherichia coli heat-labile enterotoxins (LT toxins) (4, 16). Live carrier systems include plants, bacteria, and viruses (19). Bacterial systems for heterologous antigen presentation have attracted considerable interest, but because these rely largely on live attenuated pathogens such as Salmonella and mycobacteria, considerable safety concerns remain.The gram-positive bacterium B. subtilis has been extensively studied as a model prokaryotic system with which to understand gene regulation and the transcriptional control of unicellular differentiation (6, 18). This organism is not regarded as a pathogen and is classified as a novel food that is currently being used as a probiotic for both human and animal consumption (15). The single, distinguishing feature of this microorganism is that it produces an endospore as part of its developmental life cycle when starved of nutrients. The mature spore, when released from its mother cell, can survive in a metabolically dormant ...

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

“…Bacillus subtilis strains have been engineered to express different antigens as live recombinant vaccine vehicles delivered via the oral route either as spores or vegetative cells (5,6,8,16,17,21). Previous attempts to express the B subunit of heat labile toxin (LTB), produced by enterotoxigenic E. coli (ETEC) strains, have demonstrated that orally delivered B. subtilis vaccine strains may elicit protective systemic antibody responses to a lethal toxin challenge in mice (17).…”

Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors

“…Two spore coat proteins, CotB and CotC, have been used to display tetanus toxin fragment C (TTC) of Clostridium tetani and the B subunit of the heat labile toxin (LTB) produced by some enterotoxigenic E. coli strains (ETEC) as fused proteins expressed on the surface of B. subtilis spores , Duc et al 2003a, Mauriello et al 2004 (Table I). Both antigens were expressed as C-terminal fusions with the Cot proteins, which are by themselves anchored at the outer layer of the spore coat at a number of approximately 1,000 molecules/spore , Duc et al 2003a.…”

“…Both antigens were expressed as C-terminal fusions with the Cot proteins, which are by themselves anchored at the outer layer of the spore coat at a number of approximately 1,000 molecules/spore , Duc et al 2003a. In contrast to the attempts to employ B. subtilis strains as expression systems of heterologous proteins for subunit vaccines, the genes encoding hybrid spore coat proteins were integrated into specific sites at the bacterial chromosome, which conferred stability to the expression of the recombinant genes (Duc et al 2003a, Mauriello et al 2004.…”

“…Both antigens were expressed as C-terminal fusions with the Cot proteins, which are by themselves anchored at the outer layer of the spore coat at a number of approximately 1,000 molecules/spore , Duc et al 2003a. In contrast to the attempts to employ B. subtilis strains as expression systems of heterologous proteins for subunit vaccines, the genes encoding hybrid spore coat proteins were integrated into specific sites at the bacterial chromosome, which conferred stability to the expression of the recombinant genes (Duc et al 2003a, Mauriello et al 2004. Mice orally treated with three consecutive daily doses of recombinant spores, given three times at intervals of two weeks, developed statistically significant secreted fecal (IgA) and serum (IgG) antibody levels which, at least in the case of TTC, could confer protection to mice challenged with lethal doses of the toxin (Duc et al 2003a, Mauriello et al 2004.…”

“…Such features could be attributed to the ubiquitous presence of spores in the environment and its chemical composition that is devoid of compounds known to induce inflammatory responses in mammal hosts. Thus, oral immunization regimens with recombinant B. subtilis spores require high antigen loads (above 10 10 spores/dose) and repeated immunizations (Duc et al 2003a, Mauriello et al 2004. Indeed some experimental evidences suggested that most of the local and systemic immunogenicity of recombinant B. subtilis spores is triggered during the intracellular germination and transient persistence of the nascent vegetative cell in the phagosome of phagocytic cells, as macrophages (Duc et al 2003b.…”