Towards the development of a vaccine for proliferative kidney disease

该提案将研究影响虹鳟鱼水产养殖业的一种重要疾病，即增殖性肾病（PKD），目标是在疫苗开发方面取得重大进展。这种疾病是由一种被称为粘虫的微小寄生虫引起的，这种寄生虫会在鱼类中引起严重的免疫反应，其特征是慢性肾脏病变。我们最近的工作发现，被称为苔藓虫的群体淡水无脊椎动物是感染鱼类的寄生虫的储存库。最近，我们发现温度升高会导致寄生虫在苔藓虫中增殖，释放出更多的孢子，这表明随着气候变化导致水温升高，这种疾病可能会在养殖和野生鲑鱼种群中变得更加严重。在瑞士的野生褐鳟鱼种群和挪威的野生大西洋鲑鱼种群中已经有这种影响的证据，我们知道过去几年在英格兰南部的鳟鱼养殖场，PKD的严重程度有所增加。目前，没有任何治疗方法可以预防或控制这种疾病。然而，PKD的一个重要特征是，在最初感染寄生虫后存活的鱼对随后的暴露具有抗性，因此用疫苗引发免疫系统有望成为控制这种疾病的一种方法。关键是找到一种合适的分子，可以触发保护性免疫反应。在最近的研究中，我们已经确定了一些PKD分子，其中几个具有共同的特点，在其他宿主-病原体模型中引起保护的分子。因此，在本提案中，我们将研究这些分子的有效性。我们将使用疫苗技术的最新进展，并在大多数情况下使用DNA疫苗而不是蛋白质疫苗，因为前者是筛选候选疫苗的成本效益高的方法，并且以前已被证明在鱼类中效果良好。鱼将在每年遭受这种疾病的商业虹鳟鱼养殖场接种疫苗，结果将根据存活率和引发的免疫反应进行评估。然而，我们不会假设最好的候选疫苗将在我们已经测序的基因中，因此也将采取另一种方法。第二种方法将使用从鱼类宿主以及中间宿主中分离的病原体材料，并使用分子生物学技术，我们将从这种材料中创建“基因文库”，其中包含许多可以由这种病原体表达的基因。以与上述相同的方式，我们将使用DNA疫苗方法来筛选这些基因库中的候选疫苗，但在这种情况下，将使用许多基因的“池”。用这些不同的“池”接种疫苗的鱼将如上所述进行抗病性评估，显示有益影响的批次将被细分和重新测试，最终给出少量潜在的候选分子以进行更详细的研究。由于病原体菌株变异会影响分子的分子组成，我们将对最初方法中的一些候选基因进行调查，看看它们是否不变并适用于所有菌株，或者它们是否会发生变化，最终的疫苗需要来自多个来源。通过这种方式，我们将根据我们过去在发现寄生虫基因方面的成功以及我们在分子生物学、PKD生物学和鱼类疫苗接种方面的现有专业知识，采取系统的方法开发这种疾病的疫苗。

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.