Cyanobacterial Extracellular Vesicles as Protein Carriers: Towards Fish Vaccination

Fish aquaculture faces significant economic losses from disease outbreaks. Vaccination is the most effective prevention strategy, and bacterial extracellular vesicles (EVs) show promise as vaccine platforms due to their strong immuno-stimulating properties. However, the application of EVs derived from pathogenic bacteria is limited by toxicity risks and production challenges. Alternatively, genetic engineering of non-pathogenic microorganisms is being explored to produce tailored EVs to deliver antigens and serve as carriers of therapeutic proteins. Recently, we have engineered the model cyanobacterium Synechocystis sp. PCC 6803 for the expression of the reporter green fluorescent protein (sfGFP) and its targeting to EVs. Here, taking advantage of the Synechocystis sfGFP-loaded EVs, the stability of vesicles and their cargo was evaluated in the long term when stored under different temperature conditions and after freeze-drying. The possibility of using Synechocystis EVs as a tool for eliciting specific/adaptive immune responses was assessed in European seabass, a high commercial value fish, by following the amount of total and sfGFP-specific immunoglobulins produced after immunisation through injection. Synechocystis EVs were shown to be resilient nanostructures that can induce specific immune responses in fish with additional adjuvant features. This represents a biotechnological breakthrough towards a novel antigen-carrier platform for sustainable fish-pathogen control.