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

虹鳟鱼养殖是英国水产养殖部门的重要组成部分。增生性肾脏疾病（PKD）是影响鳟鱼生产的重要疾病之一。目前还没有控制PKD的治疗方法，PKD是由一种叫做苔藓沙门氏菌的不寻常寄生虫引起的，它是一种黏液动物。该疾病通过受感染苔藓虫传播给易感鱼类，苔藓虫是生活在河流系统中的群居（无脊椎）动物。我们已经研究这种疾病很多年了，并且以前描述了在鳟鱼中引起的免疫反应，其中失调是明显的。我们已经进行了大量的序列分析，以表征两个宿主中寄生虫的所谓转录组（表达基因库）。初步的疫苗接种试验加上确定哪些基因在鳟鱼宿主中主要表达的分析，使两种新的候选疫苗（P14G8和C-39373）得以确定，以供进一步研究。由于我们最初的疫苗工作使用了DNA疫苗方法，我们建议现在转向测试基于蛋白质的两种分子疫苗，因为这有几个优点，包括可能在疫苗中添加进一步的免疫增强剂，称为佐剂。将采取两种办法；主动和被动免疫。为了进行主动免疫（目的1），在商业佐剂存在的情况下，将P14G8和C-39373蛋白单独或联合注射鳟鱼。这些蛋白质将在细菌（大肠杆菌）中使用既定的程序生产。以P14G8为例，它还将作为与鞭毛蛋白的融合蛋白制成，其中两个分子通过分子方法端到端连接，使我们能够制造单个蛋白质。我们之所以这样做，是因为鞭毛蛋白在鱼类中是一种非常有效的免疫刺激剂，因此可能比单独使用佐剂产生更好的反应。还将使用两组对照鱼，这些鱼只接受佐剂，或者不接受治疗。我们将在我们曾多次使用的两个渔场（养鱼场）进行预防PKD的评估，我们的项目伙伴将提供实物支持，以支付设施的使用、鱼/鱼的维护和讨论进展的会议费用。这些鱼最初将在一个“干净”的地方饲养两个月，以便在接种疫苗后产生免疫力，然后转移到目标养殖场，随着水温的升高，这些鱼将暴露在寄生虫中。疫苗接种对肾脏病理和PKD寄生虫负荷的影响将在试验结束时进行评估。我们对P14G8的进一步分析揭示了一些与寄生虫毒力机制有关的独特特征。我们已经生成了针对该分子的单克隆抗体（MoAb），并证明它可以在感染的肾脏切片中检测出布氏菌。奇怪的是，P14G8蛋白似乎是局部分泌的，并包围着邻近的白细胞。由于我们的免疫分析和病理反映了免疫失调，我们假设P14G8的分泌可能是阻止宿主建立有效反应的一种手段。因此，我们还将尝试使用被动免疫方法（目标2）阻断该分子，以评估这是否可以减少病理和/或使疾病产生抗性。我们已经在我们的MoAb分泌细胞系中对抗体基因进行了测序，并将利用这些信息制作可以结合P14G8的单链抗体。这种结构将被克隆到一个载体中，该载体允许蛋白质在鱼细胞中表达，然后注射到鱼体内（作为克隆的DNA），以产生预先形成的抗体。鱼将在移动到目标地点之前进行免疫接种，评估对病理和寄生虫负荷的影响，作为主动免疫接种。在方案结束时，我们计划进行讨论，以评估疫苗商业化的潜力。

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.