The mucosal surfaces of the Atlantic salmon: A site for viral entry and possible passive immunization.

In the fish farming industry, infectious diseases pose an important problem with respect to economy as well as animal welfare. To counter this problem, injection vaccines against specific diseases are frequently used. Besides, fish strains resistant to certain diseases have also been introduced. The use of vaccines however, has proven to be ineffective against a wide range of viral diseases. Moreover, injection vaccination is also expensive for the fish farmers, as each fish has to be individually vaccinated and it is stressful for the fish. It is also not suitable for small sized fish or after sea transfer. Development of alternative therapeutic methods against infectious diseases is therefore essential. Within this project, we aimed to study the possibility for passive immunization of fish as an alternative treatment. In passive immunization, antibodies towards a specific pathogen developed in a different animal are incorporated in fish feed. These antibodies may block the binding sites on the pathogen and prevent intestinal uptake of the infectious agent. It may also prevent the interaction of pathogen and fish immune cells resulting in a reduced immune reaction. Taken together, this may help reduce the extent of the infection. IPN-virus causing infectious pancreas necrosis (IPN), an important disease in the salmon industry, was our model-pathogen of interest. Single-domain antibodies (sdAb) derived from llama was our choice of antibodies as they have proven to be powerful viral neutralizers with a great therapeutic potential. These antibodies (Abs) have a significantly different profile than antibodies collected from traditional donor animals by lacking the conventional light chains. Due to their small size, they can bind to epitopes in clefts of folded proteins that are inaccessible to traditional larger antibodies and they may penetrate tissues and cells that classic antibodies cannot. The lack of a light chain in camelid antibodies also makes them less susceptible to environmental changes and they have a high tolerance to heat and pH. This in turn, makes them easier to manipulate genetically and to produce with high yields as recombinant proteins for therapeutic purposes. The project work may be divided into two phases. The first phase involved immunization, identification of specific antibodies, determining their sequence and optimization of recombinant protein. The second phase included testing the antibodies under different conditions and testing their neutralization properties. The production of antibodies with high affinity to IPNV was however not successful; although we finally managed to obtain weak binding to IPNV in ELISA by expressing the antibodies with a soluble tail. Stability of the antibodies in the intestinal environment such as gastric fluids and mucosal secretions, was thus tested using an antibody (GFP-sdAb) already characterized and with verified binding affinity for the target protein (GFP). Uptake studies over the intestinal epithelium was moreover performed using this antibody (anti-GFP). We are still considering passive immunization as a promising strategy and think that such basic studies will be of importance in this regard. As part of the project, a review paper was published in 2017 in the high regarded Journal of Immunology that will be of a more general interest with its title «Passive Immunization of Farmed Fish». A manuscript concerning the stability of llama sdAbs is further under preparation and will be submitted to an international scientific journal before the end of 2017. In addition, results concerning the construction of different recombinant sdAbs and modification of protein expression strategies are obtained, that we hope could lead to a paper in the future.

By generating knowledge on infection routes across the mucosal surfaces and by examine alternative therapeutic strategies to prevent and reduce the risk of disease outbreaks, this application meets the priorities in the call set by the Norwegian Research Council, (HAVBRUK, Tema 2 - Frisk Fisk). Infectious disease poses a serious threat to the productivity of the aquaculture industry. Disease resistant fish strains and vaccines are valuable tools to fight diseases, but knowledge on additional therapeutic s trategies will in the future be essential to hold back present and emerging diseases and keeping a healthy and sustainable industry. With this in mind, the current project will provide fundamental insight into uptake across the mucosal surface and the pot ential of passive immunization in salmonids. To examine this possibility, specialized recombinant antibodies will be developed toward infectious pancreatic necrosis virus. The antibodies will be tested for their virus neutralizing abilities in Atlantic sa lmon, but also used as a model to examine the potential of such passive immunization in general. Passive immunization of salmon can be administrated where and when there is a high risk of infection. Such immunization systems may be very helpful, particul arly during the seawater growth stages in which vaccination through injection is not possible. Passive immunization will not, by it self, give humoral immunity, but could potentially block infection or reduce the severity of disease to give the fish suffi cient time to develop its own immune response. If an outbreak is diagnosed, these antibodies targeted toward the pathogen may have the capability restrain transmission by reducing the shedding and uptake of the pathogen.