This proposal intends to apply Synthetic Biology to create a new bacterial species, Vaccinobacter, devoted to the production of multivalent, highly effective vaccines. The project originates from the evidence that Outer membrane Vesicles (OMVs) naturally produced by all Gram-negative bacteria can induce remarkable protective immunity, a property already exploited to develop anti-Neisseria vaccines now available for human use. OMV protection is mediated by the abundance of Pathogen-Associated-Molecular Patterns (PAMPs), known to play a key role in stimulating innate immunity. Moreover, OMVs can be engineered by delivering recombinant proteins to bacterial periplasm and outer membrane. Intrinsic adjuvanticity and propensity to be manipulated potentially make OMVs an ideal vaccine platform, particularly indicated when antigen combinations (for pathogens with genetic variability) and strong potentiation of immunity (for the elderly and cancer) are needed. However, full exploitation of OMVs as vaccines is prevented by: i) presence of potentially reactogenic compounds such as LPS, virulence factors, and toxins, ii) presence of several irrelevant proteins, which dilute immune responses, iii) lack of broadly applicable molecular tools to load OMVs with foreign antigens. Scope of the project is to provide novel solutions to solve these limitations and demonstrate the unique performance OMVs as vaccines by testing them on complex pathogens and cancer. Main project activities are: 1) remodelling of E. coli genome to create “Vaccinobacter”, a “living factory” of OMVs deprived of all unnecessary components but carrying the relevant immune potentiators, 2) characterization and optimization of the immune stimulatory properties of OMVs, 3) development of novel methods to incorporate foreign antigens into Vaccinobacter-derived OMVs, 4) loading of OMVs with selected pathogen- and cancer-derived antigens and demonstration of their protective efficacy in appropriate animal models.