Mapping fish CD4 T cell subsets for vaccine improvement

该项目涉及阿伯丁大学和智利圣地亚哥大学的两个专家小组之间的合作，在鱼类免疫学/鱼类疫苗接种的互补领域进行最先进的研究。水产养殖是为不断增长的世界人口提供食物的增长最快的部门之一。据估计，全世界消费的鱼中约有50%是养殖的，预计这一数字还会上升。鱼类养殖的可持续性依赖于对鱼类健康的良好管理和疾病的控制。疫苗接种是控制许多常见疾病的有效策略，目前已有许多高效的鱼类疫苗。然而，鱼类疫苗的开发在很大程度上是经验性的，基于一种配方是否有效地增加疾病后的生存挑战。从伦理和科学的角度来看，这都是不令人满意的。显然需要建立改进鱼类疫苗开发的方法。该项目将进行研究，以确定可能对未来鱼类疫苗开发至关重要的重要免疫途径。疫苗接种依赖于刺激脊椎动物的适应性免疫，在遇到病原体时具有长期记忆反应提供保护。在哺乳动物中，驱动这种反应的关键效应群是辅助性T细胞（Th）细胞，它们释放细胞间介质（细胞因子），启动抗微生物反应，包括抗体的产生。这些反应必须针对病原体类型进行调整，因为病毒、寄生虫和细胞外细菌需要不同的免疫机制来提供保护。不同的Th亚群对这些不同的病原体类型和宿主因子有不同的反应，并释放不同的细胞因子以产生最合适的反应。我们对鱼类的这些反应几乎一无所知，尽管哺乳动物中涉及的许多基因现在已经被确定，或者有可能具有同等功能的假定同源物。例如，Th细胞在其表面表达CD4，硬骨鱼中存在两种类型的这种分子，这可能会定义这些细胞。在这里，我们建议开发针对这两种CD4分子的抗体试剂，以检测、分离和表征CD4亚群（CD4-1+, CD4-2+, CD4-1+/CD4-2+）。在免疫的鱼中，我们将研究它们在体外用特定抗原再刺激后表达不同细胞因子的能力。将分析分类后的CD4细胞群。作为抗原，细菌和病毒蛋白都将用于虹鳟鱼，这是智利和英国重要的养殖鱼类。我们已经开发了许多试剂（如用于免疫基因表达分析的引物）和免疫蛋白（如重组细胞因子）用于鳟鱼，并期望反应能更普遍地代表鲑科。在初步实验之后，我们将通过分析细胞因子基因表达和CD4亚群变化，研究添加不同细胞因子和特异性抗原对后续反应的影响。接下来，我们将选择在引导这些反应中表现出最显著作用的细胞因子，以确认CD4（假定的Th）细胞的参与。这将通过在重组细胞因子存在的情况下体外抗原再刺激后，分析分选（CD4）细胞亚群中的细胞因子基因表达来完成。这些结果将大大有助于确认鱼体内Th细胞的功能，并将确定它们是否能够表达不同的细胞因子库来响应特定的抗原和细胞因子。我们预计，这些反应将在未来的疫苗开发规划中作为保护标志具有价值，有助于改进性能差的疫苗的效力，并生产针对新出现疾病的疫苗。它们还可以提供一种评估疫苗性能的替代方法，减少遭受病原体攻击的鱼的数量。

This project involves collaboration between two teams of experts at the University of Aberdeen and University of Santiago of Chile, conducting state-of-the-art research in complementary areas of fish immunology/fish vaccination. Aquaculture is one of the fastest growing sectors that provide food to the expanding world population. It is estimated that ~50% of fish consumed worldwide are farmed, and this figure is projected to rise. Sustainability of fish farming relies on good management of fish health and control of diseases. Vaccination is an effective strategy to control many common diseases and many highly efficacious fish vaccines exist. However, the development of fish vaccines has been largely empirical, based on whether a formulation is effective at increasing survival post-disease challenge. This is unsatisfactory from both ethical and scientific perspectives. There is a clear need to establish methods to improve fish vaccine development.This project will undertake studies to characterise an important immune pathway that may be of vital importance for future fish vaccine development. Vaccination relies on the stimulation of adaptive immunity in vertebrates, with long-term memory responses giving protection when encounter with a pathogen occurs. In mammals a key effector population driving such responses are T helper (Th) cells, that release intercellular mediators (cytokines), that initiate antimicrobial responses, including antibody production. These responses have to be tailored to the pathogen type, with viruses, parasites and extracellular bacteria requiring different immune mechanisms to give protection. Different Th subpopulations differentiate in response to these different pathogen types and host factors, and release different repertoires of cytokines to produce the most appropriate response. We know virtually nothing about these responses in fish, although many of the genes involved in mammals are now characterised or have putative homologues likely to have equivalent function. For example Th cells express CD4 on their surface, and two types of this molecule exist in teleost fish, that will likely define these cells. Here we propose to develop antibody reagents to the two CD4 molecules to detect, isolate and characterise the CD4 subsets (CD4-1+, CD4-2+, CD4-1+/CD4-2+). In immunised fish, we will study their ability to express different cytokine repertoires upon restimulation in vitro with specific antigen. Sorted CD4 cell populations will be analysed. As antigen, both a bacterial and viral protein will be used in rainbow trout, an important farmed fish in both Chile and the UK. We have developed many reagents (eg primers for immune gene expression analysis) and immune proteins (eg recombinant cytokines) for use in trout, and expect the responses to be representative of those in salmonids more generally. Following the initial experiments, we will study the effect of adding different cytokines together with the specific antigen on the ensuing responses, by analysing cytokine gene expression and CD4 subset variations. We will next select the cytokines showing the most marked effects on directing these responses to link back to confirming the involvement of CD4 (putative Th) cells. This will be done by analysing cytokine gene expression in the sorted (CD4) cell subsets following antigen restimulation in vitro in the presence of the recombinant cytokines. These results will go a long way towards confirming the function of Th cells in fish, and will establish if they can express different cytokine repertoires in response to specific antigen and cytokines. We anticipate these responses will be of value as markers of protection in future vaccine development programmes, helping to improve the efficacy of poorly performing vaccines, and to generate vaccines to emerging diseases. They may also provide an alternative means to evaluate vaccine performance, reducing the numbers of fish undergoing pathogen challenge.