Towards the development of a vaccine for proliferative kidney disease

Lead Research Organisation: University of Aberdeen
Department Name: Inst of Biological and Environmental Sci


This proposal will study an important disease that affects the rainbow trout aquaculture industry, proliferative kidney disease (PKD), with the objective of making significant progress towards the development of a vaccine. The disease is caused by a microscopic parasite known as a myxozoan, which causes a severe immune response in fish characterized by a chronic kidney pathology. Our recent work led to the discovery that colonial freshwater invertebrates known as bryozoans act as a reservoir of parasites infective to fish. Recently we have found that increasing temperatures cause the parasite to proliferate in bryozoans with greater numbers of released spores, suggesting that that the disease is likely to become more problematic in farmed and wild salmonid stocks as water temperatures increase due to climate change. There is already evidence for this effect in wild brown trout populations in Switzerland and wild Atlantic salmon populations in Norway and we are aware that PKD has increased in severity over the past few years in trout farms in Southern England. Currently, there are no current treatments to prevent or control the disease. However, an important characteristic of PKD is that fish that do survive an initial infection with the parasite are resistance to a subsequent exposure, and thus priming of the immune system with a vaccine is expected to be one way to control this disease. The key is to find an appropriate molecule that can trigger a protective immune response. In recent studies we have identified a number of PKD molecules, several of which have characteristics in common with molecules that elicit protection in other host-pathogen models. So in this proposal we will study the effectiveness of these molecules. We will use a recent advance in vaccine technology, and use for the most part DNA vaccines rather than protein based vaccines, since the former are a cost-effective way to screen our vaccine candidates and have been shown previously to work well in fish. Fish will be vaccinated at a commercial rainbow trout farm, that suffers from this disease every year, and the results will be assessed in terms of survival and the immune response elicited. However, we will not assume that the best vaccine candidates will be amongst the genes we have already sequenced, and so will take an alternative approach as well. The second approach will use pathogen material isolated from the fish host, as well as an intermediate host, and using molecular biology techniques we will create 'gene libraries' from this material that will contain many of the genes that can be expressed by this pathogen. In the same way as above, we will use a DNA vaccine approach to screen these gene libraries for vaccine candidates, but in this case will use 'pools' of many genes. Fish vaccinated with these different 'pools' will be assessed for disease resistance as above, and batches that show beneficial affects will be subdivided and retested, to eventually give small numbers of potential candidate molecules to look at in more detail. Since pathogen strain variation can affect the molecular composition of molecules, we will undertake a survey of some of the candidate genes from the initial approach and see if they are invariant and suitable for all strains, or whether they vary and that an eventual vaccine would require to be derived from multiple sources. In this manner, we will take a systematic approach to vaccine development for this disease, based on our past success in discovering parasite genes, and our existing expertise in molecular biology, PKD biology and fish vaccination.

Technical Summary

Proliferative kidney disease (PKD) is one of the most economically devastating diseases currently affecting the aquaculture industry in the UK. Increasing water temperatures due to climate change processes are likely to exacerbate PKD outbreaks, owing to their impact on parasite development in bryozoan populations. Currently, there are no treatments for this disease, although the possibility of vaccination exists as recovering fish are known to have resistance to re-infection. Our recent work uncovered a large resource of novel genes from the PKD parasite, several of which are homologous to or have similar molecular characteristics with known protective antigens in other parasite models. Here we will determine the protective efficacy of genes of therapeutic interest as single-gene DNA vaccines. DNA vaccine technologies have become firmly established in fish vaccinology, offering high levels of protection efficacy, particularly against viruses. The most immune-protective antigen genes in this study will also be further tested by fusing each gene to a suitable molecular adjuvant, namely a parasite-derived Hsp70 fragment and by assessing the protective efficacy of the corresponding recombinant proteins. We will also undertake an unbiased approach to the discovery of protective antigens by employing expression library immunization. Full length cDNA libraries will be generated from both fish and bryozoan-derived parasite material, and sub-libraries used for immunisation. Protective sub-libraries will be sequentially fractionated and genes present in small protective gene pools finally sequenced and characterised. Looking towards the future, we will determine the sequence diversity of protective antigens in the most divergent parasite strains, as determined by ITS-1 sequence analysis. Favourable antigens for strain-transcending vaccines will be those with very low sequence diversity and very low sequence homology to known fish genes.
Description We have taken the first steps towards the development of a vaccine to control proliferative kidney disease (PKD), probably the most problematic disease facing trout farming in the UK.

Our studies have identified a group of PKD molecules sharing characteristics with immune protective molecules in other host-parasite models. We also identified molecules possessing features of surface-expressed molecules, which may come into contact with the fish host and elicit a protective immune response. Fish were vaccinated at a commercial rainbow trout farm. A single parasite molecule elicited a very low level of protection. We attempted to improve this by vaccinating fish with the same molecules joined to a parasite gene known to be highly immunogenic in other host-pathogen models. However, we found no improvement in the effectiveness of single molecules.

In a second approach, we developed new gene libraries constructed from parasite material derived from both bryozoan and fish hosts. The bryozoan-derived parasite gene library was fractionated to produce 17 smaller gene libraries (gene pools), allowing us to determine the effectiveness of 2,500 parasite molecules in each. Two first generation gene pools (SL-10 and SL-15) were found to be effective in reducing disease pathology. Further fractionation of SL-10 and SL-15 (12,500 parasite molecules each) and subsequent testing of second generation gene pools yielded a single protective gene pool (SL-R). SL-R was subjected to further fractionation and 20 third generation gene pools (3,725 parasite molecules in total) for DNA vaccination were created. Only a single third generation gene pool (R-14) was protective against PKD. We have now sequenced the R-14 gene pool and have shortlisted those molecules, which may be conveying protection. Several parasite molecules, some of which are currently unknown in gene databases, have the potential to elicit a protective immune response as outlined earlier. Our shortlist also includes 3 parasite molecules of particular pharmacological interest.

Our new gene libraries have also proved extremely useful in enhancing or understanding of the biology of the PKD parasite. Indeed, from our sequencing efforts, we identified a parasite molecule belonging to a gene family which is only found in an animal group called the cnidarians (jellyfish, corals, and sea anemones). This gene family (minicollagens) are the fundamental building blocks of the stinging cells unique to these animals. This finding provides a link between cnidarians and the cells responsible for parasite attachment to fish hosts. In doing so, we have resolved the long disputed grouping of myxozoan parasites in the animal kingdom.

In addressing how parasite strain variation can affect the molecular composition of candidate PKD vaccine molecules, we located 5 new infected bryozoan populations ensuring sufficient parasite material for our vaccine work. We found very little strain differentiation between parasites from different geographic regions in Europe. It was not clear whether use of different genetic markers would help to resolve this or if the parasite truly has not differentiated between locations as far apart as Norway and Italy. If such lack of strain variation exists in candidate vaccine genes, there is good potential for vaccine development with broad protection to most parasite strains, at least within Europe.

Overall, we have succeeded in generating new parasite gene libraries facilitating future studies of this disease. The identification and sequencing of a protective gene pool has allowed us to shortlist parasite molecules which may be conveying immune protection. This could be addressed in a follow-on study, which could open up opportunities for commercial exploitation.
Exploitation Route Ultimately if successful, a vaccine will be available for use by trout fish farmers to protect against PKD. In addition, if successful this approach may be adopted to develop efficacious vaccines to other diseases that have proven problematic to date.
Sectors Agriculture, Food and Drink

Description A step towards a vaccine against proliferative kidney disease in trout was taken in this programme, using a novel vaccination approach called expression library immunisation. The study is incremental and as such these first steps shaped a subsequent BBSRC programme to elucidate the protective antigens individually rather than as pools of molecules. Since the ultimate goal is for a commercial vaccine, the IP issues restrict to some degree the ability to communicate the results broadly at this early stage.
First Year Of Impact 2010
Sector Agriculture, Food and Drink
Impact Types Economic