Defining mechani1sms of CD8+ T-cell mediated immunity - using an integrated longitudinal model to achieve an elusive goal.

疫苗是实施的最成功和最具成本效益的卫生干预战略之一，并在减少许多人类和动物疾病的发病率方面发挥了关键作用。然而，对于许多疾病（如艾滋病毒、疟疾、非洲猪瘟），CD8+ t细胞介导的免疫被认为对免疫有重大贡献，目前还没有可用的疫苗引起保护性CD8+ t细胞反应。病毒载体疫苗是最有希望的递送系统，特别是在模式物种中。然而，在目标人类或动物物种中，这些疫苗虽然通常具有高度免疫原性（诱导t细胞反应），但仅显示出产生保护性CD8+ t细胞的有限能力。免疫原性和保护性之间的差异表明，疫苗诱导的t细胞反应的质量参数不足，而不是数量参数不足。到目前为止，我们对这些参数的理解是有限的，这种“知识差距”阻碍了我们采用完全合理的方法来设计新的、有效的疫苗递送平台。细小蜱虫是一种由蜱传播的牛的原生动物寄生虫，在撒哈拉以南非洲的畜牧业中造成每年约6亿美元的重大经济损失。这一损失主要由小农承担，对中低收入国家的一些最脆弱人群来说可能是毁灭性的。对细小t虫的天然免疫与CD8+ t细胞反应有关，这可以在实验条件下使用“感染和治疗方法”（ITM）或自身细小t虫感染细胞系（ACL）免疫来模拟，这已用于研究和表征保护性免疫。微小t虫研究的一个主要目标是开发一种亚单位疫苗，可作为可持续的微小t虫控制规划的一部分加以部署。使用多种病毒载体（金丝雀痘、Ad、MVA）递送已知的T. parva CD8+ t细胞抗原的试验已显示出免疫原性，但对体内攻击的保护作用有限。最近的数据表明，Ad/ mva诱导的CD4+ t细胞反应缺陷是导致CD8+ t细胞功能缺陷的原因。新型病毒载体的开发和试验需要大量的时间和资源投入，并受到合适候选物的可用性的限制。虽然令人失望，但目前病毒载体诱导保护性CD8+ t细胞反应的失败可以用来理解诱导的t细胞反应与等效的保护性t细胞反应的区别。在这个项目中，我们建议使用微小t细胞免疫的“纵向”模型，在该模型中，个体的t细胞反应在顺序给予非保护性（Ad/MVA）和保护性（ITM）免疫后进行深入分析，以分析影响表型转变的参数，从而定义确定CD8+ t细胞保护功效的免疫学参数。通过促进动物内部比较，该模型比传统的队列研究具有许多优势。项目的目标是定义：1。Ad/MVA诱导的非保护性CD8+ t细胞的功能和转录组与保护性t特异性CD8+ t细胞有何不同？2. Ad/MVA异源促发诱导的CD4+ t细胞的功能和转录组与保护细小t虫感染的功能和转录组有何不同？3. CD8+ t细胞的功能能力在多大程度上依赖于CD4+ t细胞？这一信息将为了解决定CD8+ t细胞疫苗成功或失败的基本免疫学机制提供数据，并可用于为微小t虫和其他人类和兽医病原体设计/工程改进的新型疫苗载体。

Vaccines have been amongst the most successful and cost-effective health intervention strategies implemented and have played a critical role in reducing the incidence of many human and animal diseases. However, for a number of diseases (e.g. HIV, malaria, African Swine Fever) for which CD8+ T-cell mediated immunity is thought to make a substantial contribution to immunity, there are currently no available vaccines that elicit protective CD8+ T-cell responses. Viral-vectored vaccines are the delivery system that has shown most promise, especially in model species. However, in target human or animal species these vaccines, although usually highly immunogenic (induce T-cell responses), have demonstrated only a limited ability to generate protective CD8+ T-cells. The discrepancy between immunogenicity and protection indicates that qualitative, rather than quantitative, parameters of the vaccine-induced T-cell responses are deficient. As yet our understanding of what these parameters are is limited and this 'knowledge-gap' prevents a fully rational approach to design of novel, efficacious vaccine delivery platforms. Theileria parva, a tick-borne protozoan parasite of cattle, causes major economic losses in livestock farming in sub-Saharan Africa of ~$600M/yr. This loss is largely borne by small-holder farmers and can be devastating for some of the most vulnerable people in low/middle income countries. Natural immunity to T. parva is associated with CD8+ T-cell responses, which can be mimicked under experimental conditions using either an 'infection-and-treatment method' (ITM) or autologous T. parva-infected cell line (ACL) immunisation which have been used to study and characterise protective immunity. A major aim of T. parva research has been the development of a subunit vaccine that could be deployed as part of sustainable T. parva control programme. Trials using a variety of viral vectors (canarypox, Ad, MVA) to deliver known T. parva CD8+ T-cell antigens have demonstrated immunogenicity but only limited protection from in vivo challenge. Recent data has implicated defects in Ad/MVA-induced CD4+ T-cell responses as contributing to deficient CD8+ T-cell functionality.Development and trialling of novel viral vectors requires substantial investments of time and resources and is limited by the availability of suitable candidates. Although disappointing, failure of current viral vectors to induce protective CD8+ T-cell responses can be exploited to understand how the induced T-cell responses differ from equivalent but protective T-cell responses.In this project we propose to use a 'longitudinal' model of T. parva immunisation in which the T-cell responses of individuals are intensively analysed following sequential administration of a non-protective (Ad/MVA) and protective (ITM) immunisation to enable the parameters that influence the transition of phenotypes to be analysed and so define the immunological parameters that determine protective efficacy of CD8+ T-cells. By facilitating intra-animal comparisons this model has a number of advantages over conventional cohort studies. The objectives of the project are to define: 1. How does the function and transcriptome of non-protective CD8+ T-cells induced by Ad/MVA heterologous prime-boost differ from protective T. parva-specific CD8+ T-cells? 2. How does the function and transcriptome of CD4+ T-cells induced by Ad/MVA heterologous prime-boost differ from those associated with protection from T. parva infection? 3. How dependent on CD4+ T-cells is the functional competency of CD8+ T-cells? This information will provide the data to understand the fundamental immunological mechanisms that dictate CD8+ T-cell vaccine success or failure and can be used to inform the design/engineering of improved novel vaccine vectors for T. parva and other human and veterinary pathogens.