Passive and active immunisation against novel vaccine targets to protect trout against proliferative kidney disease (PKD).

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Rainbow trout farming is a key component of the UK aquaculture sector. Proliferative kidney disease (PKD) is one of the most important diseases impacting trout production. Currently no treatments exist to control PKD, which is caused by an unusual parasite called Tetracapsuloides bryosalmonae that is a Myxozoan. The disease is transmitted to susceptible fish species from infected bryozoans, which are colonial (invertebrate) animals that live in river systems. We have studied this disease for many years, and previously characterised the immune responses elicited in trout, where dysregulation is apparent. We have undertaken a lot of sequence analysis to characterise the so-called transcriptome (repertoire of expressed genes) of the parasite in both hosts. Preliminary vaccination trials coupled with analysis to determine which genes are predominantly expressed in the trout host, have allowed two novel vaccine candidates (P14G8 and C-39373) to be identified for further study. Since our initial vaccine work used a DNA vaccine approach, we propose to now switch to testing protein-based vaccines for both molecules, as this has several advantages including the possibility to add further immunopotentiating agents to the vaccines that are termed adjuvants. Two approaches will be taken; active and passive immunisation. For active immunisation (Objective 1) trout will be injected with the P14G8 and C-39373 proteins individually and combined, in the presence of a commercial adjuvant. The proteins will be produced in bacteria (E. coli), using established procedures. In the case of P14G8 it will additionally be made as a fusion protein with flagellin, where the two molecules are linked end-to-end using molecular methods to enable us to make a single protein. We will do this because flagellin is a very potent immunostimulant in fish, and so may give even better responses than with adjuvants alone. Two control groups of fish will also be used, that will receive the adjuvant only, or be left untreated. Assessment of protection against PKD will be undertaken at two field (fish farm) sites we have used on many occasions, with in-kind support from our project partners to cover the costs of access to the facilities, fish/fish maintenance, and meetings to discuss progress. The fish will be kept initially at a "clean" site for two months for immunity to develop post-vaccination and then transferred to the target farm site where the fish are exposed to the parasite as water temperatures increase. The impact of vaccination on kidney pathology and PKD parasite load will be assessed at the end of the trial. Our further analysis of P14G8 has revealed some unique characteristics in relation to virulence mechanisms of parasites. We have generated a monoclonal antibody (MoAb) to this molecule, and have shown it detects T. bryosalmonae in infected kidney sections. Curiously the P14G8 protein appears to be secreted locally and surrounds adjacent white blood cells (leucocytes). Since our immune analysis and the pathology reflects an immune dysregulation, we hypothesise that secretion of P14G8 may be a means to prevent an effective response being established in the host. Hence we will also attempt to block this molecule using a passive immunisation approach (Objective 2) to assess if this can reduce the pathology and/or give disease resistance. We have already sequenced the antibody genes in our MoAb secreting cell line, and will use this information to make a single chain antibody that can bind P14G8. This construct will be cloned into a vector that allows the protein to be expressed in fish cells, for injection into fish (as the cloned DNA) to produce the preformed antibodies. The fish will be immunised just prior to movement to the target site, with the impact on pathology and parasite load assessed as for active immunisation. At the end of the programme we have discussions planned to assess the potential for a vaccine to be commercialised.