Display of Heterologous Proteins in Bacillus Subtilis Biofilms for Enteric Immunization

One of the foremost goals in vaccine development is the design of effective, heat-stable vaccines that simplify the distribution and delivery while conferring high levels of protective immunity. Here, we describe a method for developing a live, oral vaccine that relies on the biofilm-forming properties of the spore-former bacterium Bacillus subtilis. The amyloid protein TasA is an abundant component of the extracellular matrix of the biofilms formed by B. subtilis that can be genetically fused to an antigen of interest. Spores of the recombinant strain are then prepared and applied via the oral route in an animal model. Due to the intrinsic resistance of the spores, they can bypass the stomach barrier, germinate, and subsequently colonize the gut, where they develop into biofilms, expressing the antigen of interest. We describe here the steps necessary to produce spores, immunization, and downstream analysis of the vaccine efficacy.