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

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.

No treatments exist to control PKD, a devastating disease impacting rainbow trout aquaculture. It is caused by Tetracapsuloides bryosalmonae and transmitted to fish from infected bryozoans. We have characterised the immune responses in trout, where dysregulation is apparent, and sequenced the T. bryosalmonae transcriptome from both hosts using RNAseq. Preliminary vaccination trials coupled with analysis of genes predominantly expressed in the trout host, reveal two novel vaccine candidates (P14G8 and C-39373). Our initial vaccination studies used DNA vaccines that work well in fish. We propose to now test protein-based vaccines (Objective 1), as this allows inclusion of adjuvants and gives a defined dose of protein for immunostimulation.

We have made a monoclonal anti-P14G8 that detects T. bryosalmonae in infected kidney, and shows the P14G8 protein is secreted locally and surrounds adjacent leucocytes. We hypothesise this is a means to prevent effective responses establishing, and will attempt to block P14G8 by passive immunisation (Objective 2). We have sequenced the hybridoma VH and VL genes, and will make a single chain antibody (Scab) for injection into fish to produce the preformed antibodies.

Trout will be injected with E. coli produced P14G8 and C-39373 recombinant proteins individually and combined, with a commercial adjuvant. P14G8 will also be made as a fusion protein with flagellin. Controls will be fish that receive adjuvant only or left untreated. Assessment of protection will be undertaken at two field (fish farm) sites. Fish will be kept at a "clean" site for immunity to develop post-vaccination then transferred to the target farm site where the fish are exposed to the parasite. The impact of vaccination on kidney pathology and parasite load (by qPCR) will be assessed at the end of the trial. For passive immunisation, fish will be immunised just prior to movement to the target site, with protection assessed as for active immunisation.

Farmed fish are consumed worldwide and are an important source of protein in addition to meat and poultry. The UK has a sizeable fish farming industry, primarily for rainbow trout and Atlantic salmon. However, the sustainability of the industry is not assured, with a number of disease issues in particular impacting on ambitious production targets, with a significant economic cost. If these disease issues can be controlled then improved fish health/ welfare, and increased production can be expected. The present research programme is focused on one such disease issue, namely proliferative kidney disease (PKD) in rainbow trout, considered to be one of the most economically damaging diseases for trout farming in the UK and Europe. Therefore the work has the potential to impact on the aquaculture industry, by finding a means to control an important disease, with benefits including 1) reduced mortalities, leading to 2) increased and more profitable production and 3) enhanced fish welfare. In addition, it may impact on 4) fish vaccine companies that could gain a new product for marketing and increased revenue, 5) academia, in terms of disseminating a proof-of-principle that fish vaccines against parasites can be developed, with the methodology transferable to other host-pathogen systems, and 6) the wider public, by contributing to a sustainable supply of fish for the table.

No therapeutic treatments exist for PKD, but it is known that recovered and re-exposed fish have a low parasite prevalence/ parasite intensity compared with fish exposed to PKD for the first time. These findings support the concept that fish can develop immunity to this parasite, and hence that vaccination should be possible if the appropriate antigens can be discovered. Building on past studies we have identified two vaccine candidates that will be assessed for their effectiveness to protect against PKD in the present study. The antigens will be administered singly and in combination, with different adjuvants, and the effects determined in trials at a major rainbow trout farm. Passive immunisation will also be trialled to assess whether it is possible to reduce the induced kidney pathology using this approach, since this outcome would also have benefits if complete immunity could not be established.

This project involves UK partners from academia (University of Aberdeen) and the UK trout farming industry (Test Valley Trout, Trafalgar Fisheries, British Trout Association). The partnership is crucial for evaluating (candidate) vaccine efficacy and ensures the results are relevant in the context of a trout farm environment. Positive findings will be reviewed with our partner and other relevant stakeholders, with a view to identifying opportunities to enable a vaccine to PKD to be commercialised, to gain the benefits outlined above.