Use of cell culture technology to minimize the needs for animal trials in development and production of fish vaccines

Niels Lorenzen

Prophylaxis of infectious diseases is better than cure, both in man and in husbandry animals. The latter also include aquacultured fish, where implementation of vaccines has contributed to a significant reduction in the needs for antibiotics. However, vaccine development as well as potency testing of already commercialized vaccines rely heavily on empirical vaccination and challenge (infection) trials with experimental fish.

The proposed project aims at substituting the use of live animals with cell cultures for the initial screening of vaccine components, before moving into animal experiments. Also, the project aims at establishing laboratory tests for confirmation of vaccine efficacy including batch potency testing.

Established cell cultures from different tissues of rainbow trout will be exposed to vaccine components and their response and uptake will be evaluated by gene expression and microscopical analyses, respectively.
The work will link up with previous and ongoing research projects for linking the results with data from fish trials. The project will thus by itself not require use of additional experimental animals.

The outcome of the project is expected to partly replace the use of live animals with cell cultures in initial steps of vaccine development and production. This will reduce the number of experimental animals needed within these fields. Refinement will be addressed by initial screening of vaccine components on cell cultures for potential toxic/negative side effects, hereby allowing avoidance of exposure of animals to potentially harmful elements.

Project status - december 2017

Aquaculture is a rapidly expanding industry which gradually substitutes fisheries on the decreasing wildlife fish populations as a provider of healthy animal food products for human consumption. Like other husbandry animals, farmed fish face problems with infectious diseases. Losses due to disease outbreaks and needs for treatment with antibiotics and othercompounds represent a major challenge for the growing industry. Prophylaxis is better than cure, and considerable research are invested in development of efficient vaccines for farmed fishes.

Successful vaccines against important bacterial diseases have since 1987 implicated a more that 10-fold reduction of needs for antibiotics in salmon farming in Norway. In Denmark we aim at similar results by developing more and better vaccines for aquacultured fish. However, vaccine development as well as potency testing of already commercialized vaccines rely heavily one mpirical vaccination and challenge (infection) trials with live fish.

This implies use of many of experimental fish in DK and other countries every year. In this project we aimed at substituting the use of live animals with cell cultures for the initial screening of vaccine
components. Vaccines must be able to trigger both innate and adaptive immunity, and we here applied a recombinant variant of a fish virus encoding Green Fluorescent Protein (GFP) in studies of the ability of the virus to cope with both types of immune mechanisms in cell culture.

Time course studies in cell cultures expressing interferon following inoculation with the potential vaccine adjuvant component “poly I:C” revealed that while the cells upregulated expression of key innate immune response genes and appeared resistant to the virus shortly after stimulation, the effect was temporary and extended incubation without poly I:C allowed the latent virus infection to proceed and infect all cells.

Inoculation of susceptible cell cultures with virus in the presence of a highly neutralizing monoclonal antibody demonstrated that the virus, within a single passage was able to escape from the neutralizing antibody by a point mutation in the viral glycoprotein. The results demonstrate that cell cultures can be very useful in studies of how vaccine induced immune mechanisms can interfere with a virus infection and also that it is important to take the limitations of these mechanisms into account in vaccine design. We plan to disseminate the results to other researchers and fish vaccine developers by publication in a suitable scientific journal.

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