Towards the development of a vaccine for proliferative kidney disease

该提案将研究影响虹鳟鱼养殖业的一种重要疾病，即增殖性肾病（PKD），目的是在研制疫苗方面取得重大进展。这种疾病是由一种被称为黏液虫的微小寄生虫引起的，它在鱼类中引起严重的免疫反应，其特征是慢性肾脏病理。我们最近的研究发现，被称为苔藓虫的殖民地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，温度升高会导致这种寄生虫在苔藓虫中繁殖，释放出更多的孢子，这表明，随着气候变化导致水温升高，这种疾病在养殖和野生鲑鱼种群中可能会变得更有问题。在瑞士的野生褐鳟种群和挪威的野生大西洋鲑鱼种群中已经有了这种影响的证据，我们也意识到，在过去的几年里，在英格兰南部的鳟鱼养殖场，PKD的严重程度有所增加。目前，还没有有效的治疗方法来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下来的鱼对随后的暴露具有抵抗力，因此用疫苗启动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，我们已经确定了许多PKD分子，其中一些与其他宿主-病原体模型中引起保护的分子具有共同特征。所以在这个提议中，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，并在很大程度上使用DNA疫苗而不是基于蛋白质的疫苗，因为前者是筛选我们的候选疫苗的一种具有成本效益的方法，并且以前已证明在鱼类中效果良好。每年将在遭受这种疾病的商业虹鳟鱼养殖场对鱼进行疫苗接种，并根据存活率和引起的免疫反应对结果进行评估。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，因此我们也将采取另一种方法。第二种方法将使用从鱼类宿主中分离出来的病原体材料，以及中间宿主，并使用分子生物学技术，我们将从这些材料中创建“基因库”，其中包含许多可以由该病原体表达的基因。与上述相同，我们将使用DNA疫苗方法筛选这些基因库以寻找候选疫苗，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并重新测试，最终给出少量潜在的候选分子以进行更详细的研究。由于病原体菌株的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，是否适用于所有菌株，或者它们是否不同，最终的疫苗需要从多个来源获得。通过这种方式，我们将基于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.