Defra - Department for Environment, Food and Rural Affairs.

Science Search

Science and Research Projects

Return to Science Search homepage   Return to Project List

Development of a Tuberculin Replacement Test - SE3318

Skin testing for Bovine Tuberculosis (bTB) is based on purified protein derivatives of tuberculin derived either from Mycobacterium bovis (PPD-B) alone, or, for the comparative tuberculin test (SICCT), combined with PPD from M. avium (PPD-A). PPDs are crude extracts of mycobacterial cultures with largely undefined content and active components. They are difficult to standardise with their biological activity (potency) determined with the ‘guinea pig potency test’ that is difficult to standardise. Tuberculin skin test sensitivity and/or specifcity can also be impacted by infection with, or vaccination against, Johne’s Disease (JD).
To overcome these limitations, we hypothesized that addition of further antigens to our prototype reagents composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test: DST) would increase skin test reactions to enhance assay robustness and sensitivity. In project SE3305, we developed a molecularly defined tuberculin (TRT) composed of recombinant proteins and synthetic peptides. Five additional antigens were formulated together with the three DST antigens. TRT exhibited significantly increased skin responses compared to DST, whilst maintaining 100 % specificity. We also demonstrated the compatibility of the TRT with the in vitro Interferon-gamma release blood test (IGRA). However, before the TRT can move from the research into the development space, a number of critical knowledge gaps need to be closed, which is the aim of the current proposal.
Objective 1: Confirm the sensitivity and specificity of TRT in skin and blood test in naturally M. bovis infected cattle and cattle vaccinated against JD. This will allow an appreciation of test performance in challenging testing scenarios.
Objective 2: Identify the dominant immunological active components of TRT. The fewer components that are part of a test reagent, the easier and cheaper will be its production. Therefore, using in vitro IGRA assays with Peripheral Blood Mononuclear Cells (PBMC) from M. bovis infected, uninfected and JD vaccinated animals we will establish the immune-dominance of individual TRT constituents to potentially reduce TRT complexity by removing low antigenic proteins. The output will be optimized 2nd generation TRTs, applying pai-wise test compraisons
Objective 3: Generate 2nd generation TRT formulations based on recombinant fusion proteins or synthetic peptides. Based on our experience with the DST of using either a single fusion protein, or a cocktail of long synthetic peptides, we will apply the same approaches for the TRT. Information gathered in objective 2 will also be used to design the optimal antigen composition. The outcome will be to establish TRT formations for testing in objective 4 of reduced complexity or chemical synthesis of peptides to reduce manufacture and QA processes.
Objective 4: Evaluation of the TRT formulations generated in objective 3. Skin and blood tests will be performed in relevant animal groups (M. bovis infected, vaccinated against JD) to compare these optimized formulations against the original TRT developed in SE3305. This will establish a lead TRT formulation that could be further developed towards a commercial product.
Objective 1: The outputs of this objective will be the assessment of the TRT2 skin test and IGRA performance in deliberately MAP sensitized cattle (specificity) as well as field reactor cattle (sensitivity) to benchmark responses obtained with experimental animals in SE3305. It will also provide the opportunity to re-calibrate/re-calculate the provisional cut-off value for the TRT2 skin test of > 4 mm established in project SE3305. Bio-banked PBMC from these animals will be banked and used to achieve objective 2.

Objective 2: Appreciations of specificity of individual TRT2 components in respect to MAP and an appreciation of the relative contribution each antigen makes to test sensitivity. Potentially a simplified 2nd generation TRT reagent with only the absolutely essential components, which would ease production processes and thus production costs.

Objective 3: Two 2nd generation TRT formulations for testing in objective 4, one solely composed of synthetic peptides (pepTRT), the other composed of fusion proteins (fusionTRT).

Objective 4: Biological data on the performance of the second generation TRT reagents generated in objective 3 (including refined cut-offs, and sensitivity and specificity estimates). Based on this data, a decision on which of the three formulations tested (original TRT2, fusionTRT, pep TRT) to take forward into the development space.
Time-Scale and Cost
From: 2019

To: 2021

Cost: £856,228
Contractor / Funded Organisations
APHA (Animal and Plant Health Agency)