Bacillus subtilis Spore Surface Display System Protects Recombinant Proteins from Degradation-Verified Hypothesis

Endospores of Bacillus subtilis have been used extensively as a platform for recombinant protein display for nearly two decades. Main use of the spore surface display system is generation of oral vaccines against many human and animal pathogens. Formulation of oral vaccine based on spores is an attractive approach to alternative vaccination due to the timesaving and relative easiness of production. Another advantage of such formulation is stability of presented antigens. It is assumed that the spore coat structure prevents degradation of displayed proteins by many destructive factors such as heat, proteases or stomach environment. However, there is little scientific background and substantial lack of experiments verifying this statement. In our work, we tested protective properties of spores against degradation of displayed antigens in harsh environment. We constructed B. subtilis strains producing spores presenting highly conserved long α-helix (LAH) region of the influenza A virus hemagglutinin. The constructs were obtained by fusion of LAH antigen to protein CotB or CotZ from the spore coat. We treated recombinant spores with destructive agents such as heat, protease, low pH and high-energy irradiation to test protective features of the spore coat. After treatment, spore coat extracts were analyzed by western blot to study fate of the displayed antigen. Results that we publish indicate that spore coat protects displayed antigen from degradation. This work is a strong support of hypothesis stating protective properties of spore surface display system against antigen degradation.