Chemical Inactivation of Bacillus subtilis Endospores Preserves Recombinant Protein Antigenic Properties

Recombinant Bacillus subtilis endospores are promising bacterial expression platforms for oral protein delivery, such as oral vaccines. A simple and effective spore inactivation method that preserves protein functionality, however, is needed to prevent potential shedding into the environment. This study evaluated iron or copper combined with EDTA and ethanol as sporicidal solutions for the inactivation of recombinant spores expressing the 1PR82 gene. Immunoblot and immunofluorescence (IF) assay confirmed the presence of antigenic proteins post-treatment, while electron microscopy (SEM/TEM) assessed spore morphology. Mice immunization tested immunogenicity, and fecal analysis monitored gastrointestinal persistence. Iron ethanol treatment completely inactivated the spores while maintaining recombinant protein detection using antibody-based assays. SEM/TEM revealed morphological damage, yet antigenicity was preserved, as evidenced by robust IgG responses in immunized mice. Fecal analysis showed no prolonged spore shedding, confirming effective inactivation. These findings demonstrate that iron ethanol efficiently inactivates recombinant B. subtilis spores without compromising protein antigenicity. Despite structural damage, the recombinant protein remained immunogenic, and inactivated spores posed no environmental persistence risk. This inactivation method supports the safe use of Bacillus subtilis recombinant spores for oral delivery applications, balancing inactivation efficacy with functional protein preservation. Further research could optimize this approach for clinical or industrial applications.