Development of recombinant BCG vaccine and complementary diagnostics for TB control in cattle.

由牛分枝杆菌引起的牛结核病（TB）在发达国家和印度等发展中国家仍然是一个主要问题。牛支原体感染不仅会给畜牧业造成巨大的经济损失，还可以通过气溶胶或食用受污染的乳制品从受感染的牛身上传播给人类，从而引起人畜共患结核病。在西方，牛结核病的控制是通过发现并随后杀死受感染的动物。感染的检测主要是通过结核菌素皮肤试验（PPD），将从结核杆菌（结核菌素）中提取的粗变性抗原制剂注射到动物的皮肤下，然后在几天后进行检查，此时受感染的动物在注射部位已产生强烈可见的炎症反应。这种测试既便宜又有效，但它有一个主要缺点：接种了标准结核病疫苗卡介苗（牛结核杆菌的一种减毒菌株）的动物在测试中报告呈阳性。这种交叉反应性意味着PPD试验不能用于诊断接种疫苗的动物的结核病。因此，为了保留PPD试验，不使用卡介苗疫苗来控制牛结核病，尽管它在牛身上有既定的功效。在印度，任何诊断和屠宰控制战略的另一个问题是，宰杀受感染的牛在文化上是不可接受的。PPD与卡介苗交叉反应的同样问题困扰着控制人类结核病的规划，这也是一些国家（尤其是美国）不使用卡介苗疫苗的原因。然而，最近开发了新的诊断测试，可以区分卡介苗接种和结核杆菌的自然感染。这些检测基于免疫原性蛋白，这些蛋白的基因在形成卡介苗的过程中从牛支原体基因组中自发丢失，因此可用于区分牛支原体感染和卡介苗接种（DIVA抗原）。这些测试作为基于血液的干扰素- γ释放测定（IGRA），现已广泛用于诊断人类结核病；但它们在检测牛的牛支原体感染方面不如PPD灵敏。另一个缺点是，这些测试是基于实验室的，需要从受感染的动物身上采集血液样本，在严格的时间内和温度控制的运输条件下运送到实验室，进行测试并将结果报告给现场。这使得目前的IGRA DIVA测试价格昂贵，不适合印度等技术和科学基础设施有限的国家。然而，近年来在将这些DIVA抗原应用于皮肤试验方面取得了重大进展。这些研究表明，DIVA皮肤试验是IGRA试验的一种现实可行的替代方法，其敏感性接近但尚未达到PPD的敏感性。重要的是，它们的特异性在皮肤试验中应用时，似乎优于IGRA （bbb99 %）。因此，这些研究提供了一个原则证明，即对牛结核病更具成本效益的控制策略，特别是在印度，将廉价有效的疫苗（如卡介苗）与廉价有效的皮肤试验诊断试剂结合使用，以识别和分离受感染的动物。这个项目的目的就是开发这样一个系统。该项目将有两个互补的组成部分。首先，我们将从目前的卡介苗中删除编码额外的不需要抗原的基因，有效地将牛支原体和卡介苗中均存在的非DIVA抗原转化为牛支原体中存在但卡介苗中不存在的DIVA抗原。与此同时，我们将开发一种标准化的皮肤试验试剂，其中包含现有和新的DIVA抗原（从新的卡介苗中去除的抗原）的混合物。

Bovine tuberculosis (TB) caused by Mycobacterium bovis remains a major problem in both the developed world and in developing countries such as India. As well as being a cause of huge economic loss in livestock farming, M. bovis infection can be spread to humans from infected cattle by aerosol or by consumption of contaminated dairy products to cause zoonotic tuberculosis. Control of bovine TB in the west is by detection and subsequent killing of infected animals. Detection of infection is primarily by the tuberculin skin test (PPD) in which a preparation of crude denatured antigen from the tubercle bacillus (tuberculin) is injected under the skin of the animal and is then examined several days later, by which time infected animals have generated a strong visible inflammatory reaction at the site of injection. The test is cheap and effective but it suffers from a major drawback: animals that have been vaccinated with the standard TB vaccine, BCG (an attenuated strain of the bovine TB bacillus Mycobacterium bovis), report positive in the test. This cross-reactivity means that the PPD test cannot be used to diagnose TB in vaccinated animals. So, in order to retain the PPD test, BCG vaccination is not used to control bovine tuberculosis, despite its established efficacy in cattle. An additional problem for any diagnosis and killing control strategy in India is that it is culturally unacceptable to cull infected cattle. The same problem of PPD cross-reactivity with BCG besets programmes for control of TB in humans and is the reason why BCG vaccination is not used in several countries, most notably the USA. However, new diagnostic tests have recently been developed that can distinguish between BCG vaccination and natural infection with the TB bacillus. These tests are based on immunogenic proteins whose genes were spontaneously lost from the genome of M. bovis during the development into the BCG vaccine and so can be used to Differentiate between natural Infection with M. bovis and VAccination with BCG (DIVA antigens). These tests, applied as blood-based interferon-gamma release assays (IGRA), are now widely used to diagnose TB in humans; but they are not as sensitive as PPD for detecting M. bovis infection in cattle. An additional drawback is that the tests are laboratory-based requiring blood samples to be taken from infected animals, transported to the laboratory within a tight timeline and under temperature controlled transport conditions, the test performed and result reported back to the field. This makes current IGRA DIVA tests expensive and inappropriate for countries, such as India, with limited technological and scientific infrastructure. However, over recent years significant progress has been made in applying these DIVA antigens in skin tests. These studies demonstrated that DIVA skin testing constitutes a realistic and practical alternative to IGRA tests with sensitivities approaching, but not yet reaching, those of PPD. Importantly, their specificity, when applied in skin tests, appears to be superior compared to their use in IGRA (>99 %) Therefore, these studies provided proof of principle that a much more cost-effective control strategy for bovine tuberculosis, particularly in India, would be to implement a combination of a cheap and effective vaccine (such as BCG) together with a cheap and effective skin test diagnostic reagent to identify and segregate infected animals from the rest of the herd. It is the aim of this project to develop such a system. The project will have two complementary components. First we will delete genes encoding additional dispensable antigens from the current BCG vaccine, effectively convert non-DIVA antigens present in both in M. bovis and BCG into DIVA antigens present in M. bovis but absent from BCG. In parallel, we will develop a standardized skin test reagent containing a cocktail of both current and new DIVA antigens (those removed from the new BCG vaccine).