Towards an in vitro system of predictive biomarkers of in vivo liposome efficacy

疫苗的设计目的是在不需要自然感染的情况下，在个体中诱导保护性免疫反应。它们含有“抗原”（整个病原体或病原体亚单位），需要免疫应答。佐剂是疫苗的重要组成部分，特别是那些不含活病原体的疫苗，因为佐剂可以增加所提供的抗原物质的效力。鉴于“活疫苗”由于其安全性较差，被更先进的亚单位和DNA疫苗所拒绝，因此佐剂在疫苗开发中发挥了关键作用。目前有一系列不同的佐剂可用，但脂质体（一种中空的脂质“袋”）的使用非常有吸引力，因为它们具有既具有佐剂性质又具有递送抗原的能力（例如在空心核内或在其表面）的优势。本项目旨在对脂质体进行有效的体外筛选，以确定更有效的佐剂制剂。对于每一种新的疫苗配方，许多体内试验对于确保疫苗的有效性和安全性至关重要。因此，新型脂质体制剂的产生往往是进化的，而不是革命性的，即基于已显示出一些用途的制剂的微小修改。这种渐进的方法是缓慢的，而筛选大量配方库的能力将加速开发新的和更有效的疫苗，以预防一系列重要疾病（例如结核病、艾滋病毒、疟疾和衣原体）。然而，目前，大规模筛查在动物和工时方面的成本高得令人望而却步。在这个项目中，我们将测试已经在体内测试过的脂质体配方（从有效到无效），包括具有不同物理和化学特性的配方，以及产生不同“类型”免疫反应的配方，即所谓的Th1和Th2反应。这将使我们能够在没有进一步体内研究的情况下挖掘有效和无效反应的数据。针对每种疾病产生适当“类型”的免疫反应对任何疫苗的成功都至关重要。例如，Th1反应可以防止细胞内的病原体，如病毒，因此Th2反应是不可取的。我们将在一系列体外测试中测试我们的脂质体，并通过将结果整合到“系统生物学”方法中，我们将确定体外生物活性的“指纹”，从而预测体内疗效。这将节省大量动物的使用，并将改善药物发现管道，因为我们将能够有效地筛选大型配方库，以确定进一步工作的线索。因此，该项目将加速新型脂质体和疫苗的开发。我们独特的小组体外测定已被精心挑选，是相关的免疫反应的产生。一旦注射疫苗，疫苗必须吸引并激活被称为抗原呈递细胞（APC）的关键免疫系统细胞。因此，我们将在体外评估APC在每个脂质体存在下的行为。我们将评估(A) APC向脂质体的迁移；(B)脂质体与APC的关联；(C)脂质体活化APC（通常是静止的）和(D)将体外结果与已知的体内功效联系起来。我们的方法将确定脂质体体内功效的关键体外标志物。这种“生物标记指纹”将在未来的工作中用于筛选体外制剂库，以确定可能的体内功效。这种方法将确定有效的脂质体体内制剂的关键生物标志物。这样做的净效果将是加快和提高疫苗开发管道的效率，对人类健康产生重大有益影响。

Vaccines are designed to induce protective immune responses in individuals without the need for natural infection. They contain 'antigens' (whole pathogens or sub-units of pathogens) against which an immune response is desirable. Adjuvants are an important component of vaccines, especially those that do not contain live pathogens, as adjuvants increase the efficacy of the antigenic material that is provided. Given that 'live vaccines', due to their less tolerable safety profile, are rejected in favour of more advanced sub-unit and DNA vaccines, adjuvants thus play a key role in vaccine development. There are a range of different adjuvants currently available but the use of liposomes (a hollow 'bag' of lipid) are highly attractive as they have the advantage that they are both adjuvant in nature and also have the capacity to deliver antigens (e.g. within the hollow core or on their surface). This project is directed towards the effective in vitro screening of liposomes for the identification of more effective adjuvant preparations.With every new vaccine formulation, many in vivo tests are essential to ensure efficacy and safety of the vaccine. Thus the generation of novel liposome formulations has tended to be evolutionary rather than revolutionary i.e. minor modifications based on a formulation that has shown some use. Such an incremental approach is slow and the ability to screen large libraries of formulations will speed the development of new and more effective vaccines for the prevention of a range of important diseases (e.g. TB, HIV, malaria and chlamydiae). However currently, large-scale screening is prohibitively expensive in animals and man-hours.In this project we will test liposome formulations that have already been tested in vivo (ranging from effective-ineffective) including formulations with varying physical and chemical characteristics and those that generate different 'types' of immune responses, so called Th1 and Th2 responses. This will allow us to data mine both effective and ineffective responses without further in vivo studies. Generation of the appropriate 'type' of immune response for each disease is critical to the success of any vaccine. For example, Th1 responses protect against pathogens such as viruses that live within cells and thus a Th2 response would not be desirable. We will test our liposomes in a range of in vitro tests and by integrating the results in a 'systems biology' approach, we will identify a 'fingerprint' of in vitro biological activity that is predictive of in vivo efficacy. This will save the use of very significant numbers of animals and will improve the drug-discovery pipeline, as we will then be able to efficiently screen large libraries of formulations to identify leads for further work. This project will thus speed the development of novel liposomes and vaccines.Our unique panel of in vitro assays has been carefully selected to be relevant to the generation of immune responses. Once a vaccine is injected, a vaccine must attract and activate key immune system cells known as antigen presenting cells (APC). Consequently we will assess in vitro the behavior of APC in the presence of each liposome. We will assess (A) migration of APC to liposomes; (B) association of liposomes with APC; (C) liposome-activation of APC (that are usually quiescent) and (D) correlate the in vitro results with known in vivo efficacy.Our approach will identify key in vitro markers of in vivo efficacy of liposomes. This 'biomarker fingerprint' will then be used in future work to screen libraries of formulations in vitro for likely in vivo efficacy. This approach will identify key biomarkers of effective in vivo preparations of liposomes. The net effect of this will be to speed and make more efficient the vaccine development pipeline with significant beneficial impact for human health.