Efficacy, heat stability and safety of intranasally administered Bacillus subtilis spore or vegetative cell vaccines expressing tetanus toxin fragment C

Abstract

Bacillus subtilis strains expressing tetanus toxin fragment C (TTFC) were tested as vaccine candidates against tetanus in adult mice. Mice received three intranasal (IN) exposures to 10⁹ spores or 10⁸ vegetative cells of B. subtilis expressing recombinant TTFC. Immunized mice generated protective systemic and mucosal antibodies and survived challenge with 2× LD₁₀₀ of tetanus toxin. Isotype analysis of serum antibody indicated a balanced Th1/Th2 response. Lyophilized vaccines stored at 45 °C for ≥12 months, remained effective. Immunized conventional and SCID mice remained well, and no histological changes in brain or respiratory tract were detected. Lyophilized/reconstituted B. subtilis tetanus vaccines administered IN to mice appear safe, heat-stable, and protective against lethal tetanus challenge.

Introduction

Development of vaccines that are temperature resistant and needle-free would be a welcome addition to immunization programs in developing countries, where sustaining the cold chain can be problematic and safe injection practices are often circumvented. Our own group first considered the possibility of engineering Bacillus subtilis for use as a non-invasive, heat and environmentally resistant antigen delivery system [1]. Others have subsequently pursued this idea with encouraging results [2], [3]. B. subtilis, a soil organism, has several additional characteristics of interest as a vaccine antigen delivery system. It is generally regarded as safe for humans and is readily amenable to genetic manipulation [4]. Stable constructs can be created by integration into the bacterial chromosome, making this bacterium a good candidate to express peptide vaccine antigens. In our initial report of a B. subtilis spore-based vaccine we found that oral administration to mice of spores expressing the invasin antigen of Yersinia pseudotuberculosis in the replicating vegetative stage of growth induced a systemic antibody response [1]. Mauriello et al. [5] subsequently expressed tetanus toxin fragment C (TTFC) as a fusion product with a spore coat protein. They reported that recombinant spores expressing TTFC on their surface given either orally or intranasally induced antigen-specific immune responses independent of their ability to germinate in the gastrointestinal tract. This success was mitigated by the high doses of spores needed for immunization (∼10¹⁰ spores given in six doses) and by the apparent degradation of much of the surface-displayed antigen during passage through the GI tract. An additional potential problem with this construct is that an antigen displayed on the spore surface would not be expected to have the same heat stability as the spores themselves or proteins carried within the spore.

To determine the most effective way of immunizing using the B. subtilis system, we compared strains engineered to express TTFC on the spore surface or in the vegetative cell cytoplasm and employed immunization regimens based on oral vs. intranasal (IN) inoculation with respect to efficacy, immunogenic stability and safety. The results were compared to those obtained with commercial, alum-adjuvanted, intramuscularly delivered DTaP vaccine.

IN administration is an attractive alternative to oral immunization, especially for surface-expressed antigens, since the antigen is presented directly to the nasopharyngeal immune system without any need for the spores to germinate or for germinated spores or cells to replicate. In our hands, IN administration of B. subtilis spores or vegetative cells was highly effective in immunizing mice against tetanus toxin. In their lyophilized forms, the vaccine strains maintained full protective immunogenicity for at least 12 months at 45 °C.