Transfer of a non-human primate (NHP) in vitro functional assay for the early evaluation of TB vaccine candidates and the associated immune response

结核病仍然是一个严重的全球健康威胁，目前唯一可用的卡介苗是不够的。迫切需要一种更有效的疫苗。然而，由于尚不清楚哪些免疫措施表明具有保护作用，目前正在小鼠、牛和非人灵长类动物（NHPs）等动物模型中测试新的候选疫苗。国家卫生服务提供者被认为是特别相关的，因为它们发展出最像人类的结核病形式，并且在现场使用的国家卫生服务提供者正在增加。动物接种疫苗，然后感染结核病的病原体结核分枝杆菌（M.tb），以测试疫苗是否具有保护作用。这一过程的严重程度被归类为中度，这意味着动物可能会经历“短期中度疼痛、痛苦或痛苦，或长期轻度疼痛、痛苦或痛苦……或健康或一般状况的中度损害"随着疾病的发展，动物可能出现体重减轻、发烧和呼吸窘迫，如果不及时治疗，最终会死于肺功能不全；需要人道安乐死。在过去的6年里，Tanner博士领导了一项体外或“试管”试验的发展，该试验通过接种疫苗的动物或人的血液或细胞来测量结核分枝杆菌（或来自同一家族的其他细菌）的控制，而不是感染动物或人自己。这大大降低了动物疫苗试验的严重程度，属于3r的“细化”类别。它还允许在一组动物的一组细胞中测试多种条件或免疫反应，并允许在进行测试之前早期降低候选疫苗的选择，从而减少所需的动物数量。从长期来看，这项工作旨在证明该检测是一种有意义的保护措施，允许连接到人类样本的使用（通过与人类功效的比较来证明这在逻辑上或伦理上都是不可能的）。使用人类细胞的能力可以在很大程度上取代在结核病疫苗效力测试中使用动物。该项目的主要目标是成功地将NHP MGIT分析转移到NHP结核病疫苗测试领域的两个主要实验室：英国公共卫生部（PHE）和荷兰的生物医学灵长类动物研究中心（BPRC）。这些实验室每年使用约100-150只猕猴进行结核分枝杆菌攻击实验，并且能力不断增加，实施MGIT检测可以大大减少对这一程序的需求。转移将涉及四个阶段：标准化（确保所有实验室以相同的方式进行分析），协调（确保同一实验室内重复和实验室之间相同样品的结果一致），验证（确保结果与体内感染后的保护措施相比具有生物学意义），最后是探索阶段，以调查潜在的免疫机制。先前在PHE和BPRC进行的研究中储存的样本已分配给该项目，这意味着不需要进一步的动物实验。通过成功地转移检测，这些小组将能够将其应用到他们未来的疫苗研究中，从而产生相当大的当地影响。此外，该项目将产生数据，证明MGIT分析是可重复和可转移的概念验证，从而增加全世界其他结核病研究小组的兴趣。有证据表明，这种测定与体内结核分枝杆菌感染的保护密切相关，这将进一步证实生物学有效性，并给科学家和疫苗开发人员带来信心。

Tuberculosis (TB) remains a serious global health threat and the only currently available vaccine, BCG, is inadequate. A more effective vaccine is urgently needed. However, as it is unclear which measures of immunity indicate protection, new vaccine candidates are currently tested in animal models such as mice, cattle and non-human primates (NHPs). NHPs are considered particularly relevant as they develop the most human-like form of TB, and the use of NHPs in the field is increasing. Animals are vaccinated and then infected with the causative agent of TB, Mycobacterium tuberculosis (M.tb), to test whether the vaccine is protective. This procedure is classified as Moderate in severity, meaning that the animals are likely to experience "short term moderate pain, suffering or distress or long-lasting mild pain, suffering or distress... or moderate impairment of the well-being or general condition". As disease progresses, animals may experience loss of body weight, fever and respiratory distress and if left untreated will eventually die of pulmonary insufficiency; necessitating humane euthanasia. For the past 6 years, Dr Tanner has led the development of an in vitro, or "test tube", assay which measures control of M.tb (or other bacteria from the same family as a surrogate) by blood or cells from a vaccinated animal or person, rather than infecting the animal or person themselves. This downgrades the severity of vaccine testing in animals considerably, falling under the 'refinement' category of the 3Rs. It also allows the testing of multiple conditions or immune responses in a set of cells from a single group of animals, and allows early down-selection of vaccine candidates going forward to testing, thereby reducing the numbers of animals required. In the longer-term, this work aims to demonstrate that the assay is a meaningful measure of protection, allowing bridging to the use of human samples (as comparison with efficacy in humans to prove this is not logistically or ethically possible). Ability to use human cells could largely replace the use of animals in TB vaccine efficacy testing.The primary aim of this project is to successfully transfer the NHP MGIT assay to two major laboratories in the field of NHP TB vaccine testing: Public Health England (PHE) and the Biomedical Primate Research Centre (BPRC) in the Netherlands. These laboratories use ~100-150 macaques in M.tb challenge experiments each year with capacity increasing, and implementation of the MGIT assay could drastically reduce the requirement for this procedure. The transfer will involve four phases: standardisation (ensuring that all laboratories are performing the assay in the same way), harmonisation (ensuring that results from the same samples are concordant between replicates within a laboratory and between laboratories), validation (ensuring that results are biologically meaningful compared to measures of protection following in vivo infection), and finally an exploratory phase to investigate the underlying mechanisms of immunity. Stored samples from studies previously conducted at PHE and BPRC have been allocated for this project, meaning that no further animal experiments will be necessary. By successfully transferring the assay, these groups will be able to implement it into their future vaccine studies, having considerable local impact. Furthermore, the project will yield data demonstrating proof-of-concept that the MGIT assay is reproducible and transferable, thus increasing interest from additional TB research groups worldwide. Evidence that this assay correlates strongly with protection from in vivo M.tb infection will further confirm biological validity and give confidence to scientists and vaccine developers.