22-ICRAD Call 2 - Improving the diagnosis of tuberculosis in domestic ruminants through the use of new antigens and test platforms

Bovine tuberculosis (BTB) presents a significant challenge to animal and public health globally. BTB is also a significant cause of zoonotic TB (zTB), mainly in developing/low and middle income countries (LMIC) as highlighted in the recently published roadmap to zTB eradication jointly produced by WHO, WOAH and the International Union against TB & Lung Disease (UTLD). Their One Health approach acknowledged that TB eradication in animals will impact zTB in humans. Eradication could be accelerated by improved diagnostic tests that overcome limitations associated with Purified Protein Derivative (PPD) tuberculin: limited performance, standardisation, vaccine-interference. These could be overcome by defined antigens. PPDs are used both in tuberculin skin tests and blood interferon-gamma release assays (IGRA). More sensitive blood test platforms that complement the IGRA test could further enhance diagnostic performance. BTB tests primarily target
cellular immunity, yet serological tests can also be useful for additional case detection. Thus, our proposal aims to address these constraints to optimise test performance in an inter-connected manner. We will conduct field validation for test sensitivity and specificity of the defined antigen formulation DST-F (fusion protein of ESAT6, CFP10, Rv3615c) in domestic ruminants (cattle, goats, buffalos) in different epidemiological settings (WP1, skin test, IGRA). Environmental exposure to mycobacteria in the farms will be also evaluated. We will develop a multi-cytokine platform (MCP) to enhance test performance beyond the current IGRA, (WP2) and also develop novel antigen capture and lateral flow assays (WP3). The performance of these innovative tests will be assessed with samples generated in WP1. WP4 will critically assess data generated in WP1-3 and provide recommendations for the optimal combinations of test platforms to accelerate BTB eradication.

One critical factor that affects performance of TB diagnostic tests is the quality of Tuberculin PPD. PPDs are poorly characterized and biological activity may differ substantially between manufacturers, despite the requirements for potency evaluation. Thus, research efforts have lead to the identification of novel antigens derived from M. bovis that can be produced as synthetic peptides or recombinant proteins. The most advanced defined antigens for IT and IGRA are based on the antigens ESAT6, CFP10, and Rv3615c. These antigens, presented as fusion protein DST-F, are currently under trial in GB cattle for field safety and specificity, whilst the determination of field sensitivity in cattle, and performance in other species, is a knowledge gap that this proposal will close. These antigens also elicit other cytokine responses in addition to IFN-gamma; thus to maximise the utility of blood-based tests we propose to develop a multi-cytokine platform to be used with DST-F to maximise test sensitivity compared to IGRA. Additionally, a variety of tests have been developed that rely on the detection of antibodies (Ab) to MTBC antigens. In general, these assays have shown lower sensitivity than the CMI assays. Nevertheless, new platforms based on polyprotein fusion antigens or protein complexes such as P22 have been developed and show promising results for improved TB specific-Ab detection. Cost-effective Ab-based diagnostic platforms have merit, particularly for animal species not subject to mandatory eradication schemes. Here we will study the performance of P22 antigen as a serology-target to complement CMI tests. We will develop and validate novel diagnostic platforms based on P22 such as an ELISA for Ag detection and a lateral flow rapid diagnostic test. Environmental exposure to mycobacteria in the farms will also be evaluated using a novel system based on direct PCR in order to investigate potential interference with diagnosis.