{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:03:28Z","timestamp":1785409408899,"version":"3.56.0"},"reference-count":22,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T00:00:00Z","timestamp":1759881600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T00:00:00Z","timestamp":1759881600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100000277","name":"Department for Environment, Food and Rural Affairs, UK Government","doi-asserted-by":"publisher","award":["SE3318"],"award-info":[{"award-number":["SE3318"]}],"id":[{"id":"10.13039\/501100000277","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000277","name":"Department for Environment, Food and Rural Affairs, UK Government","doi-asserted-by":"publisher","award":["SE3332"],"award-info":[{"award-number":["SE3332"]}],"id":[{"id":"10.13039\/501100000277","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000277","name":"Department for Environment, Food and Rural Affairs, UK Government","doi-asserted-by":"publisher","award":["SE3339"],"award-info":[{"award-number":["SE3339"]}],"id":[{"id":"10.13039\/501100000277","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Clin Microbiol"],"published-print":{"date-parts":[[2025,10,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Bovine tuberculosis is an infectious livestock disease of global economic and zoonotic importance. Surveillance programs are largely dependent on the skin test that utilizes purified protein derivatives (PPDs) of tuberculin. PPDs suffer from a number of limitations regarding their production, characterization, and performance. Recently, we developed a molecularly defined tuberculin (MDT) that overcame these limitations. The MDT formulation comprises eight antigens, initially presented as seven recombinant proteins (ESAT6, CFP10, Rv3615c, Rv3020c, Rv1789, Rv3478, and Rv3810) and one 20-synthetic peptide pool representing Rv3616c; the recombinant Rv3616c could not be produced. In this paper, we describe the steps taken to simplify the MDT formulation. First, seven of the eight proteins were formulated as three recombinant fusion proteins. Second, the non-immunogenic regions of Rv3616c were identified, using overlapping peptides to probe T-cells from infected cattle. A novel variant, Rv3616c-S4, was designed and then formulated (i) as a stand-alone protein in the MDT-S4 formulation and (ii) fused to Rv1789 for use in the MDT-F formulation. These novel MDT formulations gave comparable signal strength to PPD-B in experimentally infected animals. The MDT-F, comprising only fusion proteins, performed as well as the comparative cervical tuberculin skin test, in terms of relative sensitivity (&gt;2 mm; 96%, 95% CI 80\u2013100,\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 24) and specificity (&gt;2 mm; 100%, 95% CI 89%\u2013100%,\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 30). In conclusion, we have developed a novel, simplified MDT formulation that has significantly advanced our efforts toward licensure and which has the potential to replace the PPDs developed in the 1930s.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>Bovine tuberculosis is an infectious livestock disease of global economic and zoonotic importance. Surveillance programs are largely dependent on the skin test, which utilizes tuberculins. These are crude protein extracts of live bacterial cultures, which suffer from a number of limitations regarding their characterization, standardization, production, and performance. We aim to develop a skin test reagent composed of eight defined antigens that could replace the tuberculins. This study describes the refinement of this \u201cmolecularly defined tuberculin\u201d (MDT) reagent into a fusion protein formulation comprising all eight antigens. The MDT is well defined, easily standardized, and delivers good test performance in experimentally infected, non-infected cattle and cattle sensitized to Johne\u2019s disease. The fusion protein formulation is an important step on the developmental pathway to a registered and marketable product.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jcm.00552-25","type":"journal-article","created":{"date-parts":[[2025,8,29]],"date-time":"2025-08-29T13:52:23Z","timestamp":1756475543000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Optimization of a molecularly defined tuberculin formulation: recombinant fusion proteins and epitope surgery"],"prefix":"10.1128","volume":"63","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-7287-0440","authenticated-orcid":true,"given":"Sonya","family":"Middleton","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0378g3743","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Bacteriology, Animal and Plant Health Agency","place":["Addlestone, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mahavir","family":"Singh","sequence":"additional","affiliation":[{"name":"Lionex Diagnostics and Therapeutics GmH","place":["Braunschweig, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Coad","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0378g3743","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Bacteriology, Animal and Plant Health Agency","place":["Addlestone, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Si","family":"Palmer","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0378g3743","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Bacteriology, Animal and Plant Health Agency","place":["Addlestone, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tom","family":"Holder","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0378g3743","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Bacteriology, Animal and Plant Health Agency","place":["Addlestone, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabine","family":"Steinbach","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0378g3743","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Bacteriology, Animal and Plant Health Agency","place":["Addlestone, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebecca","family":"Hardiman","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0378g3743","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Bacteriology, Animal and Plant Health Agency","place":["Addlestone, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"H. Martin","family":"Vordermeier","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0378g3743","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Bacteriology, Animal and Plant Health Agency","place":["Addlestone, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gareth J.","family":"Jones","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0378g3743","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Bacteriology, Animal and Plant Health Agency","place":["Addlestone, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_5_2_2","unstructured":"World Health Organisation. 2017. Roadmap for zoonotic tuberculosis. 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