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Updated: May 16, 2026
Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus
Published on: March 8, 2012
Safety, tolerability, and concordance with interferon-γ release assays of a recombinant ESAT6-MPT64 skin test: a
Xichao Ou1, Guan Liu2, Baozhen Peng1
1National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention (Chinese Academy of Preventive Medicine), Beijing, China.
Background:
Accurate and cost-effective screening for Mycobacterium tuberculosis (Mtb) infection remains a global challenge. While Interferon-γ Release Assays (IGRAs) offer high specificity, their widespread deployment is hindered by high costs and technical complexity. Conversely, the traditional Tuberculin Skin Test (TST) lacks specificity due to BCG cross-reactivity. Emerging recombinant skin tests predominantly rely on the ESAT6-CFP10 antigen combination. To potentially broaden the antigenic repertoire and enhance diagnostic sensitivity, we developed a novel recombinant fusion protein incorporating MPT64 (from Region of Difference 2, RD2) alongside ESAT-6. This study represents the first-in-human evaluation of the ESAT6-MPT64 (EM) skin test.
Methods:
This single-center, randomized, open-label, dose-escalation Phase 1 clinical trial (Registration: ChiCTR2500112887) enrolled 60 participants, comprising 30 healthy controls and 30 patients with active pulmonary tuberculosis (TB). Participants were stratified and randomized into low-, medium-, and high-dose cohorts (n = 10 per cohort/group). The primary endpoint was safety and tolerability. Secondary endpoints included diagnostic performance (sensitivity and specificity) and concordance with the T-SPOT.TB assay (IGRA).
Results:
The EM skin test demonstrated an excellent safety profile. Adverse events were predominantly mild (Grade 1-2), transient, and self-limiting, with no serious adverse events (SAEs) related to the investigational product reported. In terms of diagnostic performance, the test exhibited robust immunogenicity in active TB patients, achieving a peak sensitivity of 87.0% at 48 hours and maintaining 82.6% at 72 hours post-injection. In healthy controls, the test showed high specificity with a low rate of non-specific reactions (13.3%). Receiver Operating Characteristic (ROC) analysis indicated high diagnostic accuracy, with Area Under the Curve (AUC) values exceeding 0.80 across all dose groups at both 48- and 72-hour reading windows. Furthermore, the EM skin test demonstrated substantial concordance (κ > 0.60) with IGRA results, confirming that the specific DTH response is distinct from BCG vaccination background.
Conclusion:
The recombinant EM skin test is safe, well-tolerated, and demonstrates preliminary diagnostic accuracy comparable to IGRAs. By successfully validating the translational utility of the MPT64 (RD2) antigen in a human cohort, this study provides a strong proof-of-concept for the EM skin test as a scalable, specific, and cost-effective immunodiagnostic tool.
