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

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).

Control of TB in the UK is by detection of infected animals with the tuberculin skin test and culling. This is unacceptable in India. It is currently not possible to use BCG vaccine to control bovine tuberculosis because of cross-reactivity with the tuberculin test that is used to diagnose TB in cattle. This is because BCG is an attenuated strain of Mycobacterium bovis, and tuberculin is also prepared from M. bovis. IGRA-based diagnostic tests have developed for detection of TB that utilizes antigens deleted in BCG but these are not sensitive enough for bovine TB diagnosis. The key insight behind this proposal is that DIVA-based diagnostic tests and BCG-based vaccines need not be limited by the genotype of BCG strain. The first aim of this project will therefore be to develop a dcBCG deleted in additional immunodominant antigens. This will be accomplished by Tn-seq studies of persistence of BCG in cattle to identify genes that are dispensable for persistence and thereby vaccine efficacy. The deletion of BCG antigens will also be informed by proteomics analysis of tuberculin and extensive studies of immunogenicity of M. bovis antigens performed by AHVLA. We will then delete the dispensable genes from the BCG genome to generate a dcBCG strain. In parallel, we will develop an extended DIVA skin test (exDIVA) based on the current optimal set of DIVA antigens but supplemented with additional antigens deleted from dcBCG. The combination of dcBCG plus exDIVA will provide an effective vaccine for bovine tuberculosis together with a complementary cheap and effective diagnostic skin test that will be appropriate for developed countries such as the UK but particularly for developing countries such as India that are unable to implement a detection and cull control strategy. Application of this novel approach has the potential to revolutionize control of bovine TB across the world.

Bovine TB, caused by Mycobacterium bovis, is the biggest threat to the livestock industry in GB. Its incidence in cattle herds has steadily risen since the mid-1980s and the current control regime costs the taxpayer around £100 million a year. India ranks second in cattle population across the world with an estimated 199 million cattle with a very high rate of bovine TB. Although bovine TB control could be implemented through a programme of early detection and culling, this would be unacceptable in India due to socio-economic and cultural reasons.

The potential impacts of the project are:
- Development of an effective BCG-based vaccine vaccine and associated diagnostic test (DIVA) against bovine tuberculosis for use in:
o National cattle TB control programmes - initially focused on the UK and India, but potentially other countries worldwide where bovine TB is proving difficult to control.
- Establishment of novel research technologies for veterinary vaccine development in cattle, which, by improving knowledge of host-pathogen interactions, can contribute to vaccine development against other pathogens.
- One-health implications as the vaccines and diagnostic reagents could also be used to protect humans against tuberculosis.
- Defined 'tuberculins': Even in the absence of a vaccination-based control strategy, the defined tuberculins built around the antigens discovered in this project could overcome the production, QC and specificity limitations of conventional tuberculin PPD.

The research will be of great interest to academic scientists working in the areas of infectious diseases and vaccine development (particularly tuberculosis). Infectious disease is of course an extremely active area of research in the UK, India and worldwide. Tuberculosis research has been one of the active areas in infectious disease research in recent years. The specific area of research of the project is development of new vaccines and diagnostics for bovine tuberculosis, but the research will also be of interest to researchers working on human tuberculosis.

The research will also be of interest to vaccine and diagnostic test manufacturers who may be able to exploit this research to develop new vaccines and diagnostic to control bovine TB.