A reverse vaccinology approach to a bTB vaccine

牛结核病（bTB）是牛被牛分枝杆菌感染的结果。这种细菌在自然界中分布广泛，因为它也感染许多其他野生动物物种，因此，野生动物感染充当了细菌的储存库，周期性地传播到家畜身上。这样做的后果是双重的。首先，必须扑杀bTB阳性的牛，这对农民及其维持畜群的能力产生连锁反应。其次，结核分枝杆菌对人类感染构成威胁，主要是通过食用受污染的牛奶。今天，个人感染结核分枝杆菌极为罕见，但威胁仍然存在，由于这些原因，结核分枝杆菌感染是不受欢迎的，需要得到控制，或者最好是根除。自20世纪90年代中期的一次历史回顾以来，獾已被确定为将bTB传播给家畜的主要途径之一，这反过来又导致人们试图通过扑杀獾来打破这一传播途径。这些措施只取得了有限的成功，并引起了公众的极大关注，因此传播途径的物理断裂基本上没有改变。另一种方法是接受bTB在环境中广泛传播的事实，但通过以前的疫苗接种来预防牛感染。然而，目前还没有有效的牛bTB疫苗，迫切需要开发这种疫苗的新方法。人们普遍认为，疫苗的作用是通过在靶动物中产生抗体反应来阻止细菌建立初始感染。这些抗体是所有哺乳动物免疫系统的正常产物，通常会与细菌的外部结合，从而防止细菌与牛细胞（通常是肺上皮细胞）结合。由此可见，bTB的保护性成分，即产生保护性抗体反应的成分，存在于细菌表面的外部。细菌表面有许多成分，其中任何一种成分，或者是许多成分的组合，细菌基因组编码的蛋白质，对于有效免疫的发展可能是必不可少的。然而，究竟需要哪些目前尚不清楚。在这项研究中，我们建议生产bTB的所有表面成分，并批量测试它们诱导有效免疫反应的能力。我们建议在牛身上做这项工作，以便对我们的测试疫苗的反应是典型的，如果最终疫苗在典型的牛群中使用，将会发现。我们的工作可分为三个相关部分。首先，我们将从牛分枝杆菌细菌中识别出所有在生物体表面发现的蛋白质，并以安全有效的方式生产每一种蛋白质。我们的初步计算表明，可能需要数百种这样的蛋白质才能找到有效免疫所必需的少数蛋白质。其次，我们将使用我们的表面蛋白质作为牛的测试疫苗，为了使这一过程有效，我们将使用混合蛋白质进行这项工作，以便使用最少数量的牛。免疫接种后，我们将从牛身上采集血液样本，并测试它们预防牛支原体感染的能力。最后，我们将研究保护机制，以及我们确定的各个组成部分如何协同工作，为牛提供有效的免疫屏障。我们的方法是详尽的，但它有可能在bTB疫苗发现计划下划清界限，并确定最终有效疫苗生产的最佳候选组合。

Bovine tuberculosis, bTB, is the result of the infection of cattle by the bacterium Mycobacterium bovis. The bacterium is distributed widely in nature as it also infects many other wildlife species and as a result of this, wildlife infection acts as a reservoir for the bacterium which periodically get across into domestic cattle. The consequence of this is twofold. First, cattle that are bTB positive must be culled with a knock-on effect on the farmer and his ability to maintain a herd. Second, bTB is a threat for human infection, primarily via the consumption of contaminated milk. Today bTB infection of individuals is extremely rare, but the threat remains and for these reasons bTB infection is unwelcome and needs to be controlled or, preferably, eradicated. Since a historical review in the mid-1990s, the badger has been identified as one of the major routes of transmission of bTB to domestic cattle and this, in turn, has led to attempts to break this transmission route via badger culling. These have met with only limited success and immense public concern leaving the physical breaking of the transmission route mostly unaltered. An alternative approach is to accept that bTB circulates widely in the environment but to prevent cattle infection by previous vaccination. At the present time however an effective vaccine for bTB in cattle is not available and new methods to develop such a vaccine are urgently needed. It is generally accepted that vaccines function by generating an antibody response in the target animal which prevents the bacterium from establishing the initial infection. These antibodies, which are a normal product of the immune system of all mammals, generally bind to the outside of the bacterium and so prevent it from binding to cattle cells, usually the epithelial cells of the lung. It follows that the protective components of bTB, that is, the components that will generate the antibody response that is protective, are to be found on the outside of the bacterium surface. Bacteria have many components on their surface and any, or perhaps a combination of many, of these components, proteins encoded by the bacterium genome, could be essential for the development of effective immunity. However exactly which are required is currently unknown. In this research, we propose to produce all of the surface components of bTB and to test them in batches for their ability to induce an effective immune response. We propose to do this work in cattle so that the response measured to our test vaccines is typical of what will be found if an eventual vaccine is used in typical herds. Our work breaks down into three related components. Firstly, we will identify all of those proteins from the M. bovis bacterium that are to be found on the surface of the organism and produce each of them in a safe and efficient manner. Our initial calculations suggest that several hundred such proteins may be required in order to find the few that are necessary for effective immunity. Secondly, we will use our surface proteins as test vaccines in cattle and to make this process efficient we will carry out this work with mixes of proteins so that the least number of cattle has to be used. Following the immunizations we will take blood samples from the cattle and test them for the ability to prevent M. bovis infection. Finally, we will examine the mechanism of protection and how the individual components so we have identified work together to provide the cattle with an effective barrier of immunity. Our approach is exhaustive but it has the potential to draw a line under the vaccine discovery program for bTB and to identify the best mix of candidates for eventual effective vaccine production.