Toxicity study of SARS-CoV-2 Spike protein expressed on Bacillus subtilis spores.

BACKGROUND: The SARS-CoV-2 spike protein is the primary antigen used in most COVID-19 vaccines. To improve protection, especially at mucosal surfaces, a robust and safe delivery system capable of inducing both systemic and mucosal immunity is needed. Bacillus subtilis spores offer a promising platform due to their stability, safety, and capacity for protein display. A study to evaluate the potential acute oral toxicity of recombinant B. subtilis spores expressing SARS-CoV-2 spike proteins was undertaken. RESULTS: B. subtilis spore was used to express the SARS-CoV-2 S1 domain spike proteins during the bacterial sporulation. No in vitro cytotoxicity was observed when the spores were used to treat cells. BALB/c mice were given a single oral dose (5 × 10(11) CFU/ml) over three consecutive days and monitored for 14 days to assess acute toxicity. BALB/c mice fed with up to 3 × 10(11) CFU/mouse of the spores showed no adverse effects. A gross necropsy and histology of the treated mice's vital organs, kidneys, liver, heart, lungs, and spleen revealed no pathological alterations, and the biochemical parameters remained within normal ranges. CONCLUSION: These findings suggest that the SARS-CoV-2 S1 domain spike proteins expressed on B. subtilis spores is well tolerated in mice when administered orally at a high dose. Findings from this study, hence, support further investigation of the recombinant bacterial spores expressing SARS-CoV-2 S1 domain spike proteins as an oral vaccine candidate against SARS-CoV-2 infection.