Developing bovine immune organoids for the screening of vaccine candidates

一系列疾病通常影响英国和世界各地的牛，包括牛乳腺炎、布鲁氏菌病、蓝舌病、牛病毒性腹泻、口蹄疫、约翰氏病和牛结核病。这些对动物健康、粮食安全和经济都有重大影响。疫苗接种被广泛认为是最有效的控制策略，但在大多数情况下，疫苗仍需要开发或改进，需要进行筛选研究，所有获得许可的疫苗都需要定期进行批量测试。目前的体外模型未能以令人满意的方式捕获评估抗原特异性疫苗免疫原性所需的适应性免疫应答的复杂特征，并且啮齿动物是一种较差的模型，因为与牛相比，它们的免疫系统存在关键差异。因此，在体内牛的实验是常用的，这是伦理，后勤和经济costing.We建议开发牛类器官的基础上，次级淋巴器官作为一种新的工具，用于筛选牛疫苗，以取代使用牛（和啮齿动物）在早期疫苗的开发和疫苗批测试。类器官是自组织的3D组织，模拟器官的关键功能，结构和生物复杂性。牛类器官已被开发用于牛的其他几个解剖位置，包括乳腺和肠道，表明可行性。免疫类器官最近在人类中被报道，具有测量对抗原、佐剂和疫苗的初级和记忆反应的能力。我们的初步数据表明，从冷冻保存的牛淋巴结材料中生长类器官是可能的，预期的细胞再聚集和延长培养期的良好存活率。我们的目标是优化，验证和实施牛免疫类器官模型，使用牛结核病作为与动植物卫生署合作的概念验证的范例。项目合作伙伴丽莎Wagar从欧文大学，加州，开创了人类免疫类器官模型，并将提供动手培训和专业知识。对于优化阶段，我们将从未接种和接种BCG的牛中获得次级淋巴组织，并使用该组织来确定最合适的组织类型和测定条件，包括组分的组成和浓度以及随时间推移的反应动力学。我们还将在多个水平上评估重现性，然后通过证明模型可以反映体内研究的免疫原性读数来验证模型。对此的方法将是三重的：i）将文献中先前进行的研究中由Ad 85 A牛TB疫苗在体内诱导的免疫应答与在我们的类器官模型中诱导的免疫应答进行比较; ii）将文献中先前进行的研究中由一组疫苗佐剂诱导的免疫应答与在我们的类器官模型中诱导的免疫应答进行比较;和iii）平行使用体内牛研究和我们的免疫类器官模型直接比较由四种新的牛TB疫苗候选物诱导的免疫应答。为了证明该模型适用于目的，我们将实施该模型来筛选一组扩大的牛结核病候选疫苗。最后，我们将通过积极传播方法、协议和成果，通过支持培训和向相关最终用户实验室转让技术，在当地和全球建立3R遗产;牛免疫类器官的优点包括在早期牛疫苗筛选和批量测试中替代牛和啮齿动物的使用的明显3R益处，提供高通量和易处理的模型以加速牛疫苗开发的科学益处、成本效益、可转移性（包括向资源有限的环境的可转移性）以及用于一系列牛疾病以及在疫苗开发中重要的其他有蹄类动物物种的适应性。

A range of diseases commonly affect cattle in the UK and worldwide including bovine mastitis, brucellosis, bluetongue, bovine viral diarrhoea, foot and mouth disease, Johne's disease and bovine TB. These have major implications for animal health, food security and the economy. Vaccination is widely considered the most effective control strategy, but in most cases vaccines still need to be developed or improved, necessitating screening studies, and all licenced vaccines need to be regularly batch-tested. Current in vitro models fail to capture the complex features of the adaptive immune response required to assess antigen-specific vaccine immunogenicity in a satisfactory way, and rodents are a poor model due to key differences in their immune systems compared with cattle. Thus in vivo cattle experiments are commonly used, which are ethically, logistically and economically costly.We propose to develop bovine organoids based on secondary lymphoid organs as a new tool for screening bovine vaccines to replace the use of cattle (and rodents) in early vaccine development and vaccine batch-testing. Organoids are self-organised 3D tissue that mimic the key functional, structural and biological complexity of an organ. Bovine organoids have been developed for several other anatomical locations in cattle including the mammary glands and intestine, indicating feasibility. Immune organoids have recently been reported in humans, with demonstrated ability to measure primary and memory responses to antigens, adjuvants and vaccines. Our preliminary data shows that it is possible to grow organoids from cryopreserved bovine lymph node material, with the expected reaggregation of cells and good survival over an extended culture period.We aim to optimise, validate and implement the bovine immune organoid model using bovine TB as an exemplar for proof-of-concept in collaboration with the Animal and Plant Health Agency. Project Partner Lisa Wagar from the University of Irvine, California, pioneered the human immune organoid model and will provide hands-on training and expertise. For the optimisation phase, we will obtain secondary lymphoid tissue from naïve and BCG vaccinated cattle, and use this to identify the most appropriate tissue type and assay conditions including composition and concentrations of the components as well as the kinetic of response over time. We will also assess reproducibility at multiple levels.We will then validate the model by demonstrating that it can mirror immunogenicity read-outs from in vivo studies. The approach to this will be three-fold: i) comparing immune responses induced by the Ad85A bovine TB vaccine in vivo in previously-conducted studies in the literature with those induced in our organoid model; ii) comparing immune responses induced by a panel of vaccine adjuvants in a previously-conducted study in the literature with those induced in our organoid model; and iii) directly comparing immune responses induced by four novel bovine TB vaccine candidates using an in vivo cattle study and our immune organoid model in parallel. To demonstrate that the model is fit for purpose, we will then implement it to screen an expanded panel of bovine TB vaccine candidates. Finally, we will establish a 3Rs legacy locally and globally by actively disseminating methods, protocols and results; by supporting training and tech-transfer to relevant end-user laboratories; and by generating a small-scale bovine lymphoid tissue bank.The advantages of bovine immune organoids include the clear 3Rs benefits in replacing the use of cattle and rodents in early bovine vaccine screening and batch-testing, the scientific benefits in offering a high-throughput and tractable model to expedite bovine vaccine development, cost-effectiveness, transferability including to resource-limited settings, and adaptability for use across a range of bovine diseases as well as other ungulate species of importance in vaccine development.