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

针对新疫苗靶标的被动和主动免疫，以保护鳟鱼免受增殖性肾病（PKD）的侵害。

• 批准号: BB/S004076/1
• 负责人: Chris Secombes
• 金额: $ 20.27万
• 依托单位: University of Aberdeen
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS004076%2F1
• 关键词: Passive; active; immunisation; against; novel

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.

彩虹鳟鱼种植是英国水产养殖部门的关键组成部分。增殖性肾脏疾病（PKD）是影响鳟鱼产生的最重要的疾病之一。目前尚无控制PKD的治疗方法，这是由一种称为Tetracapsuloides bryosalosalmonae的异常寄生虫引起的，即粘液果。该疾病是从感染的桥梁的易感鱼类传播的，该疾病是生活在河流系统中的殖民地（无脊椎动物）动物。我们已经研究了这种疾病多年了，并以前表征了鳟鱼中引起的免疫反应，而鳟鱼则显而易见。我们已经进行了大量序列分析，以表征两个宿主中寄生虫的所谓转录组（表达基因的曲目）。初步疫苗接种试验以及分析以确定哪些基因在鳟鱼宿主中主要表达，已允许确定两名新型候选疫苗（P14G8和C-39373）进行进一步研究。由于我们最初的疫苗工作使用了DNA疫苗方法，因此我们现在建议改用两个分子的基于蛋白质的疫苗，因为这具有多个优点，包括可能将进一步的免疫构剂添加到称为辅助剂的疫苗中。将采取两种方法；主动和被动免疫。为了进行主动免疫（目标1）鳟鱼将在存在商业佐剂的情况下分别注入P14G8和C-39373蛋白。蛋白质将使用既定的程序在细菌（大肠杆菌）中产生。在p14g8的情况下，它将作为与鞭毛蛋白的融合蛋白一起制成，其中两个分子使用分子方法端到端连接，以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，因此比单独使用佐剂可以产生更好的反应。还将使用两个对照组的鱼类，仅接收佐剂或未经处理。对PKD的保护评估将在我们多次使用的两个领域（养鱼场）站点进行，并在我们的项目合作伙伴的实物支持下，以支付获得设施，鱼/鱼类维护和会议的费用，以讨论进度。该鱼最初将在“干净”地点保存两个月，以进行疫苗接种后的免疫力，然后转移到目标农场地点，随着水温的升高，鱼会暴露于寄生虫。疫苗接种对肾脏病理学和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与寄生虫的毒力机制有关的一些独特特征。我们已经对该分子产生了一种单克隆抗体（MOAB），并表明它检测到被感染的肾脏切片中的苔藓乳绿os虫。奇怪的是，p14g8蛋白似乎是局部分泌的，周围环绕着邻近的白细胞（白细胞）。由于我们的免疫分析和病理反映了免疫失调，因此我们假设P14G8的分泌可能是防止宿主中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）来阻止该分子，以评估这是否可以减少病理和/或给予抗病性。我们已经对MoAb分泌细胞系中的抗体基因进行了测序，并将使用此信息来制作可以结合P14G8的单链抗体。该构建体将被克隆到载体中，该构建体允许在鱼类细胞中表达蛋白质，将其注入鱼（作为克隆的DNA）以产生预制的抗体。在移动到目标部位之前，将对鱼进行免疫，并对病理和寄生虫负荷产生影响，以评估为主动免疫。在计划结束时，我们计划进行讨论，以评估疫苗被商业化的潜力。

项目成果

Comparative transcriptomics and host-specific parasite gene expression profiles inform on drivers of proliferative kidney disease.

• DOI: 10.1038/s41598-020-77881-7
• 发表时间: 2021-01-25
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Faber M;Shaw S;Yoon S;de Paiva Alves E;Wang B;Qi Z;Okamura B;Hartikainen H;Secombes CJ;Holland JW
• 通讯作者: Holland JW