Towards the development of a vaccine for proliferative kidney disease

该提案将研究影响虹鲑鱼水产养殖业的一种重要疾病--增殖性肾病(PKD)，目的是在疫苗开发方面取得重大进展。这种疾病是由一种名为粘虫的微小寄生虫引起的，这种寄生虫会在鱼类体内引起严重的免疫反应，其特征是慢性肾脏病理。我们最近的工作导致发现，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宿主。最近我们发现，温度升高会导致寄生虫在苔藓虫中繁殖，释放的孢子数量更多，这表明随着气候变化导致水温上升，这种疾病在养殖和野生鲑鱼种群中可能会变得更有问题。在瑞士的野生褐鲑鱼种群和挪威的野生大西洋鲑鱼种群中已经有证据表明这种影响，我们意识到过去几年来，在英格兰南部的鲑鱼养殖场，PKD的严重程度有所增加。目前，还没有预防或控制这种疾病的治疗方法。然而，PKD的一个重要特征是，在最初感染寄生虫的情况下幸存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。关键是找到一种能够触发保护性免疫反应的合适分子。在最近的研究中，我们已经确定了一些PKD分子，其中一些分子具有在其他宿主-病原体模型中诱导保护的分子的共同特征。因此，在这项提议中，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，因为前者是一种具有成本效益的方法来筛选我们的候选疫苗，并且以前已被证明在鱼类中效果良好。鱼将在每年都患有这种疾病的商业彩虹鲑鱼养殖场接种疫苗，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会出现在我们已经测序的基因中，因此也会采取另一种方法。第二种方法将使用从鱼类宿主分离的病原体材料以及中间宿主，利用分子生物学技术，我们将从这种材料中创建“基因库”，其中将包含这种病原体可以表达的许多基因。与上面一样，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，将使用许多基因库。用这些不同的“池”接种的鱼将接受如上所述的抗病评估，显示有益影响的批次将被细分和重新测试，最终提供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，我们将从最初的方法开始对一些候选基因进行调查，看看它们是否不变，是否适合所有菌株，或者它们是否不同，最终的疫苗需要从多种来源获得。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，采取系统的方法来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.