Development of Recombinant Bacillus subtilis Spore Vaccines Based on Thymine Auxotrophy as a Biocontainment Strategy

Bacillus subtilis displaying heterologous antigens show promising efficacy for mucosal immunisation against various viral and bacterial pathogens. In this work, THY-X-CISE system was used to engineer thymine auxotrophic B. subtilis strains to express immunogenic antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Helicobacter pylori on their spore surface. This system ensures containment of genetically modified (GM) Bacillus spores by inducing cell death upon exogenous thymine depletion. It also eliminates the need for antibiotic resistance gene markers, relying solely on thymine auxotrophy for positive selection.
The immunogenicity and efficacy of thymine auxotrophic spores against SARS-CoV-2 and H. pylori were evaluated, along with their ability to induce mucosal immune responses. In murine experiments, a prime-mucosal spore boost strategy using thymine auxotrophic spores expressing SARS-CoV-2 antigens evoked antigen-specific mucosal
IgA and is potentially protective, as shown in a hamster model of SARS-CoV-2 infection. Additionally, oral immunisations with thymine auxotrophic spores displaying H. pylori antigens induced mucosal responses and significantly reduced H. pylori colonisation in
mice following challenge.
In summary, the THY-X-CISE spore vaccine platform is attractive due to its potential for biological containment, rapid and cost-efficient vaccine production, and heat stability. Utilised as a mucosal booster or prophylactic vaccine, spore vaccines could benefit in
addressing current and future emerging diseases, enhancing both systemic and mucosal immunity across various viral and bacterial diseases.