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在发达国家和印度等发展中国家仍然是一个主要问题。除了在畜牧业中造成巨大的经济损失外，牛分枝杆菌感染还可以通过气雾剂或通过食用受污染的乳制品传播给人类，从而导致人畜共患结核病。在西方，牛结核病的控制是通过检测和随后杀死受感染的动物来实现的。感染的检测主要是通过结核菌素皮肤试验(PPD)，即将来自结核杆菌的粗变性抗原(结核菌素)注射到动物的皮肤下，几天后进行检查，到那时，受感染的动物已经在注射部位产生了强烈的可见炎症反应。这项测试既便宜又有效，但它有一个重大缺陷：接种了标准结核病疫苗BCG(牛结核杆菌牛分枝杆菌的减毒株)的动物在测试中报告呈阳性。这种交叉反应意味着PPD检测不能用来诊断接种疫苗的动物中的结核病。因此，为了保留PPD检测，卡介苗接种不被用于控制牛结核病，尽管它在牛身上已被证实有效。在印度，任何诊断和扑杀控制策略的另一个问题是，扑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相同问题困扰着人类结核病控制计划，这也是为什么几个国家没有使用卡介苗接种的原因，最明显的是美国。然而，最近开发了新的诊断测试，可以区分卡介苗接种和自然感染结核杆菌。这些测试是基于免疫原性蛋白，这些蛋白的基因在发展成为卡介苗的过程中自发从牛分枝杆菌基因组中丢失，因此可以用于区分自然感染牛分枝杆菌和接种卡介苗(DIVA抗原)。这些测试被用作基于血液的干扰素-伽马释放分析(IGRA)，现在被广泛用于诊断人类结核病；但它们在检测牛的牛分枝杆菌感染方面没有PPD那么灵敏。另一个缺点是，这些检测是以实验室为基础的，要求从受感染的动物身上采集血液样本，在严格的时间线内运输到实验室，并在温度控制的运输条件下进行测试，并将结果报告给现场。这使得目前的IGRA DIVA测试成本高昂，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，在皮肤试验中应用这些天后抗原方面取得了重大进展。这些研究表明，DIVA皮肤测试是IGRA测试的一种现实和实用的替代方法，其敏感性接近但尚未达到PPD的敏感性。重要的是，当它们应用于皮肤测试时，它们的特异性似乎优于在IGRA(&gt；99%)中的使用。因此，这些研究提供了原则证据，证明了更具成本效益的牛结核病控制策略将是实施廉价有效的疫苗(如卡介苗)和廉价有效的皮肤测试诊断试剂的组合，以识别受感染的动物并将其与牛群的其余部分隔离。本项目的目的就是开发这样一个系统。该项目将有两个相辅相成的组成部分。首先，我们将从目前的卡介苗疫苗中删除编码额外可有可无抗原的基因，有效地将牛分枝杆菌和卡介苗中都存在的非DIVA抗原转化为存在于牛分枝杆菌中但不存在于卡介苗中的DIVA抗原。与此同时，我们将开发一种标准化的皮肤测试试剂，其中包含现有的和新的DIVA抗原(从新的卡介苗疫苗中移除的抗原)的鸡尾酒。

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).