GreenOralVax: uma nova plataforma sustentável, amiga do ambiente e à escala nano para entrega de antigénios e vacinação oral de peixes em aquacultura

The MAIN OBJECTIVE of GreenOralVax is to DEVELOP EFFECTIVE AND SAFE ORAL VACCINES FOR THE IMMUNIZATION OF THE EUROPEAN SEABASS (ESB), by IMPLEMENTING EXTRACELLULAR VESICLES (EVs) FROM CYANOBACTERIA (CB) AS AN ANTIGEN DELIVERY SYSTEM. For its implementation, GreenOralVax’s SCIENTIFIC GOALS are the following: 1. TO ENGINEER CB FOR ANTIGEN EXPRESSION AND PACKAGING IN EVs Members of GreenOralVax have shown that it is possible to express heterologous proteins in CB and load them in EVs [1,2]. Based on literature searches and on our own results, a total of 12 different antigens from relevant ESB pathogens have been selected to be expressed in the model cyanobacterium Synechocystis sp. PCC6803 (S6803, see Fig.1 in Annex 1: Additional material). 2. TO DETERMINE THE BIOCHEMICAL COMPOSITION OF CYANOBACTERIAL EVs Cyanobacterial EVs will only become valuable tools for biotechnology if their biochemical composition is characterized in detail. However, no information is available on the composition of S6803 EVs. Therefore, a comprehensive analysis of the protein and lipid content of S6803 EVs must be carried out. This will identify what is packaged in EVs and better understand how S6803 adjusts the molecular composition of EVs when loaded with heterologous proteins. 3. TO CULTIVATE ENGINEERED CB IN PILOT SCALE BIOREACTORS AND ISOLATE ANTIGEN-LOADED EVs One of the challenges regarding biotechnology of photoautotrophic microorganisms relates to their cultivation in large scale. In spite of the good EV production capacity shown by engineered S6803 [3, WO-2023153949-A1], large amounts of culture and of customized EVs will still be needed for fish immunization. GreenOralVax will subcontract a national company, A4F, well-experienced in CB cultivation and downstream processing in pilot scale (up to 1000 L), with whom we will closely collaborate (see Supporting letter in Annex 1: Additional material). 4. TO IMMUNIZE ESB WITH ANTIGEN-LOADED EVs AND CHALLENGE GreenOralVax will generate EVs as antigen delivery vehicles. Additionally, the cyanobacterial biomass also has high value. CB expressing the heterologous antigen accumulate the produced protein intracellularly, prior to loading into EVs [2]. Therefore, once mixed with fish feed, the biomass can also work as an antigen delivery agent through the oral route. This has been experimentally tested with success with recombinant microalgae [4,5]. This way, the generated biomass will be an asset to further potentiate fish immune responses. 5. TO IDENTIFY LOCAL IMMUNE-RELATED RESPONSES OF VACCINATED ESB GreenOralVax wants to contribute to increase the fundamental knowledge regarding intestinal immune responses in fish. There is a consensus among experts of oral vaccination that antigen uptake in teleosts takes place in the hindgut, the site where immune induction is initiated locally for antigens taken up from the intestine [4]. However, the cell types, molecular elements, and even immune organs involved are yet to be definitively elucidated. Resulting from its implementation, other GreenOralVax’s objectives also include generating relevant INTELLECTUAL PROPERTY; creating JOBS; TRAINING students; DISSEMINATING the use of Aquaculture and nature-based solutions to address a major societal challenge, namely securing good quality food for all, following a One-Health approach; and ELEVATING THE RESEARCH INSTITUTES’ PROFILES, both nationally & internationally.