NKp46+CD3+ T-Cells As A Novel Target For Vaccines Against bovine TB

牛结核病（bTB）是影响英国养牛业的最重要疾病。目前的“检测和屠宰”政策未能限制bTB的传播，这导致了对未来应实施哪些策略以改善对这种疾病的控制的审查。BCG是唯一获得许可的bTB疫苗，具有可变和不可预测的保护水平，并且不符合DIVA标准，因此不能用于当前的疾病控制计划。因此，有必要开发疫苗，赋予更可靠的免疫力，并可以与bTB现场监测。此类疫苗的开发需要更好地理解i）有助于保护性免疫的免疫应答的组分和ii）如何利用免疫应答来区分接种疫苗的动物和Mb感染的动物。虽然已经确定抗TB的保护性免疫是细胞介导的，但精确的保护性免疫机制仍不明确。有证据表明，常规肽-MHCII限制性CD 4 + T细胞应答对于免疫是关键的，并且对于最佳抗TB免疫，还需要肽-MHCI限制性CD 8 + T细胞。在人类中，如果由非常规脂质特异性CD 1限制性T细胞组成，则大部分细胞抗TB应答;这些细胞表现出的抗分枝杆菌效应子功能的范围表明它们也有助于宿主防御TB。因此，已经提出脂质可以构成新的抗原，其可以用于增强下一代TB疫苗的免疫原性和功效。脂质是有吸引力的疫苗候选物，因为它们i）激活先天性样和适应性T细胞应答，ii）可以具有佐剂性质，并且iii）由非多态性CD 1基因呈递，因此（与MHC限制性肽不同）在远交群体中具有实用性。另外，有些脂质对PPD无免疫反应，可作为DIVA试剂。虽然牛脂质特异性T细胞反应已在bTB中报道，但关于其在抗bTB免疫中的作用的知识可以忽略不计。我们实验室最近进行的研究已经鉴定出一种新的非常规NKp 46 + CD 3+牛T细胞群体，并证明这些细胞的一个亚群对PIM（一组来自M.在一定比例的Mb感染动物中，在该项目中，我们建议通过在BCG免疫/Mb激发研究期间分析NKp 46 + CD 3 + T细胞来获得关于bTB中脂质特异性T细胞应答的信息。我们还将进行研究，以确定抗原性结核脂质的范围，并确定哪些是免疫原性的，从而有助于新的有效的bTB疫苗。该项目的目标是：1。定量并跟踪BCG接种后和随后暴露于M后的NKp 46 + CD 3 + T细胞应答。牛这将有助于确定NKp 46 + CD 3 + T细胞是否作为先天和/或适应性效应T细胞发挥作用，以及BCG免疫是否形成在Mb攻击期间回忆的记忆群体。检查NKp 46 + CD 3 + T细胞识别分枝杆菌的功能。这将定义NKp 46 + CD 3 + T细胞在抗TB免疫中的作用。来自目标1和2的数据也将用于检查NKp 46 + CD 3 + T细胞应答是否与BCG免疫赋予的抗bTB保护相关。鉴定可用于产生NKp 46 + CD 3+（和其他）牛T细胞应答的其他分枝杆菌脂质。很可能多种TB脂质在牛中具有抗原性，并且一些脂质特异性T细胞可能与NKp 46 + CD 3 + T细胞亚群一起存在。这项研究将扩大对bTB脂质抗原库的了解（并确定是否有任何可用于DIVA应用）。鉴定哪种分枝杆菌脂质在体内引起NKp 46 + CD 3 + T细胞应答。这将确定哪些脂质在体内具有免疫原性，因此是真正的疫苗候选物。

Bovine tuberculosis (bTB) is the most important disease affecting the UK cattle industry. Failure of the current 'test-and-slaughter' policy to constrain the spread of bTB has led to a review of what strategies should be implemented in future to improve the control of this disease. BCG, the only licensed bTB vaccine, exhibits variable and unpredictable levels of protection and is not DIVA compliant so can't be used in current disease control programmes. Consequently, there is a need to develop vaccines that confer more reliable immunity and can be integrated with bTB field surveillance. Development of such vaccines requires a better understanding of i) the components of the immune response that contribute to protective immunity and ii) how immune responses can be exploited to discriminate between vaccinated and Mb-infected animals. Although it is well established that protective immunity against TB is cell mediated, the precise protective immunological mechanisms are still undefined. Evidence indicates that conventional peptide-MHCII restricted CD4+ T cell responses are critical for immunity and that for optimal anti-TB immunity peptide-MHCI restricted CD8+ T-cells are also required. In humans a large proportion of the cellular anti-TB response if composed of non-conventional lipid-specific CD1-restricted T-cells; the range of anti-mycobacterial effector functions exhibited by these cells indicates that they also contribute to host defence against TB. Consequently, it has been proposed that lipids may constitute novel antigens that could be used to enhance the immunogenicity and efficacy of next-generation TB vaccines. Lipids are attractive vaccine candidates as they i) activate both innate-like and adaptive T-cell responses, ii) can have adjuvant properties and iii) are presented by non-polymorphic CD1 genes and therefore (unlike MHC-restricted peptides) have utility across an outbred population. In addition, some lipids don't induce responses against PPD, so could be used as DIVA reagents. Although cattle lipid-specific T-cell responses have been reported in bTB, knowledge of their role in anti-bTB immunity is negligible. Recent studies conducted by our laboratories have identified a novel population of non-conventional NKp46+CD3+ bovine T-cells and demonstrated that a subset of these cells respond to PIM, a group of lipids from M. bovis in a proportion of Mb-infected animals. In this project we propose to gain information on lipid-specific T-cell responses in bTB by analysing NKp46+CD3+ T-cells during a BCG-immunisation/Mb-challenge study. We will also perform studies to identify the range of antigenic TB lipids and determine which are immunogenic and so could contribute to new effective bTB vaccines. The objectives of the project are to:1. Quantify and track the NKp46+CD3+ T-cell responses following BCG vaccination and subsequent exposure to M. bovis. This will help determine if NKp46+CD3+ T-cells are functioning as innate and/or adaptive effector T-cells and if BCG immunisation forms a memory population recalled during Mb challenge.2. Examine the function of NKp46+CD3+ T-cells recognising mycobacteria. This will define the role of NKp46+CD3+ T-cells in anti-TB immunity. Data from objectives 1 and 2 will also be used to examine if NKp46+CD3+ T-cell responses correlate with anti-bTB protection conferred by BCG immunisation.3. Identify additional mycobacterial lipids that could be used to generate NKp46+CD3+ (and other) bovine T-cell responses. It is likely that multiple TB lipids will be antigenic in cattle and that some lipid-specific T-cells may lie out-with the NKp46+CD3+ T-cell subset. This study will expand the knowledge of the bTB lipid antigen repertoire (and also determine if any could be exploited for DIVA applications).4. Identify which mycobacterial lipids elicit NKp46+CD3+ T-cell responses in vivo. This will establish which lipids are immunogenic in vivo and are therefore genuine vaccine candidates.