MucoProtect. Novel bio-mimicking nanocarriers for oral delivery of mucosal vaccines - protection at the first line of defence.

Vaccines play a crucial role in the aquaculture industry’s overall strategy to combat infectious diseases. As fish inhabit aquatic environments, most pathogens are likely to invade through mucosal routes such as gills, skin, fin bases, and the gut. Mucosal vaccines that can elicit the most effective protective responses at these surfaces are thus essential for providing immunological defense against invading microbial agents, particularly during the initial stages of infection. Despite these potential benefits, there are only a few commercially available fish oral vaccines to date. The main goal of MucoProtect II is to demonstrate the effectiveness of our new alginate particle-based delivery platform (established in MucoProtect I) in delivering a test protein antigen to Atlantic salmon and generating a significant antibody response. Ultimately, our aim is to develop an improved antigen delivery system that can be incorporated into feeds and used as effective oral vaccines for mass immunization of farmed fish against various fish pathogens (viruses, bacteria, and parasites, including salmon lice). Oral vaccines offer notable advantages over standard oil-based injection vaccines used in aquaculture, as they allow for easier administration of immunogenic antigens without taking the fish out of the water. This practicality extends to vaccinating fish at earlier life stages and providing booster vaccines to growing fish in the sea. As of November 2023, MucoProtect nano- and microparticles containing bovine serum albumin (BSA) as a test/model protein antigen have been successfully produced with no apparent toxic effects in cell cultures and fish. A bioplex assay has also been developed for this project as a more sensitive method for measuring induced BSA antibodies in fish, in addition to standard ELISA assays used for either detecting antibodies against BSA or the presence of BSA itself. In the initial stages of this project, we observed that our MucoProtect particle preparation could induce significant titers of BSA-specific antibodies following systemic (IP injection) and mucosal (anal intubation) administration routes. However, when the same MucoProtect particle was delivered via oral intubation, no detectable antibody response was observed in our experimental fish. Similarly, no significant response was observed in our first oral vaccination attempt with the same MucoProtect particle preparation incorporated into feeds via extrusion method. Based on these preliminary in vivo trials, we hypothesize that, although the MucoProtect particle preparation itself was immunogenic, the release of BSA may have occurred too early, leading to its degradation by gastrointestinal (GI) enzymes before it could be absorbed/taken up by relevant immune cells in the salmon intestine. Consequently, three different versions of ‘slower-release’ model antigen encapsulation using MucoProtect particles were prepared, and the amount of BSA released in Atlantic salmon GI tract over time after feeding was measured for this new batch of MucoProtect oral vaccine feeds. Encouragingly, we detected significant amounts of BSA via ELISA assay in both the mid- and hindgut of sampled fish up to 48h post-feeding. This confirms that substantial quantities of our model antigen can reach the target site for uptake (second segment of the mid-intestine), potentially initiating the necessary gut-associated mucosal responses. Consistent with this, a marked increase in BSA immunoglobulins was detected via bioplex assay at Week 6 and 8 in serum samples from fish that were boosted’ every three weeks with the same oral vaccine feed, albeit at levels relatively lower compared to that previously observed with high-dose IP injection. Nevertheless, these promising results underscore the potential use of our current technology platform as an antigen carrier in the development of fish oral vaccines. Analysis of fourth fish feeding trial is currently underway, comparing antibody responses against BSA in the serum, and if possible, the gut of Atlantic salmon following oral vaccine feeding of different MucoProtect particle preparation (uncoated vs coated, nanoparticle vs microparticle) at various feeding intervals, and using different antigen doses (i.e., BSA concentration in the feeds and the estimated amount of BSA received by fish depending on the feeding scheme). The results from this trial will provide us with an improved/optimized MucoProtect vaccination feeding scheme, which can then be subsequently applied in the design of oral vaccines targeting various fish pathogens and other relevant parasites.

In parallel with the growth of aquaculture, infectious diseases have become an increasingly greater problem and currently represent the most significant constraint on sustainable aquaculture worldwide. For the salmon farming industry, the salmon lice challenge is currently the most important tasks to solve. One of the key areas of success in managing fish health in aquaculture has been the development of vaccines. However, for many diseases including salmon louse, vaccines are not available, and the farming industry keeps facing new challenges. Fish are continuously exposed to microorganisms in the aquatic environment. The mucosal surfaces lining the body form a physical barrier equipped with multiple dynamic and inducible immune effector mechanism crucial for the defence against environmental pathogens. Novel vaccine strategies that target these inductive sites properly are needed. Also, as antigens may not always evoke a sufficient immune response due to their nature, the path by which the vaccine is delivered and its auxiliary components are critical factors for the success of immunization. In MucoProtect, SINTEF and the Norwegian Veterinary Institute join advanced drug delivery technology with immune potentiating strategies to develop a novel and versatile vaccine delivery system customized for oral delivery of vaccines to fish: 1) New tailor-made and low-cost materials with inherent adjuvants will be developed and used to 2) produce nanoparticles specifically targeting key initiators of the immune response to prime the mucosal barriers to resist infection. If successful, MucoProtect will offer 3) new and more effective tools for preventing and controlling salmon lice that will increase the sustainability of the industry. MucoProtect will also 4) provide increased knowledge on central aspects of the mucosal immune system of Atlantic salmon and establish novel assays for assessing the effects of vaccines, both essential for future vaccine development. The main goal of MucoProtect-II will be to demonstrate that the new alginate particle-based delivery platform developed in MucoProtect-I can increase the efficacy of newly identified salmon lice antigen in Atlantic salmon. MucoProtect-II will have two dominant research questions: ? Can antigen in alginate particles, following (feed-based) oral administration, induce the production of antigen-specific antibodies in blood and mucus ? Can LPS via its immunogenicity act as immune stimulant in an oral particle-based vaccine, ie act as adjuvant? The project will use advanced equipment and methodology developed in MucoProtect-I to produce and characterize a range of alginate particles with variation in size, LPS content, (pH dependent) stability, antigen capacity and more. The project will develop new in vitro and ex vivo methods and models to test, among other things, stability of particles under different conditions, leakage of antigens and mucus penetration of particles of different sizes. The alginate particles will then be formulated for oral delivery (feed). Finally, the project will use vaccine trials as the critical yardstick for success: Can the antigen in the alginate particles induce specific antibodies after oral administration?