A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ. This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Guérin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood. The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development

Thermostability of IFN-gamma and IP-10 release assays for latent infection with Mycobacterium tuberculosis: A TBnet study

INTRODUCTION:Interferon-γ (IFN-γ) inducible protein 10kD (IP-10) and IFN-γ release assays (IGRAs) are immunodiagnostic tests aiming to identify the presence of specific cellular immune responses, interpreted as markers for latent infection with Mycobacterium tuberculosis. Incubation at higher temperatures could affect IFN-γ and IP-10 responsiveness in order to improve the performance of IP-10 release assays and IGRAs. AIM:The aim of this study was to assess the robustness of whole blood based IP-10 release assay and IGRAs and the effect of hyper-thermic incubation (39 °C) on the diagnostic accuracy of IP-10 release assay and IGRAs. RESULTS:We included 65 patients with confirmed pulmonary tuberculosis and 160 healthy controls from 6 European centres collaborating in the TBnet. In patients, IP-10 responses increased 1.07 (IQR 0.90-1.36) fold and IFN-γ responses decreased 0.88 (IQR 0.57-1.02) fold, with 39 °C compared to 37 °C incubation temperature. At 37 °C IGRA sensitivity was 85% and IP-10 sensitivity was 82%, whereas specificity was 97% for both tests (p > 0.8). These minor changes observed as a result of hyper-thermic incubation were not sufficient to impact IGRA and IP-10 release assay test performance. CONCLUSION:The performance of IGRA and IP-10 release assays is robust despite variations in the incubation temperature between 37 °C and 39 °C

Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6.

There is a need for an improved vaccine for tuberculosis. ESAT-6 is a cardinal vaccine antigen with unique properties and is included in several vaccine candidates in development. ESAT-6 is also the core antigen in the IFN-γ release assays (IGRA) used to diagnose latent infection, rendering IGRA tests unspecific after vaccination. This challenge has prompted the development of a companion diagnostic for ESAT-6 based vaccines, an ESAT-6 free IGRA. We screened a panel of seven potential new diagnostic antigens not recognized in BCG vaccinated individuals. Three highly recognized antigens EspC, EspF and Rv2348c were identified and combined with CFP10 in an ESAT-6 free antigen cocktail. The cocktail was prepared in a field-friendly format, lyophilized with heparin in ready-to-use vacutainer tubes. The diagnostic performance of the ESAT-6 free IGRA was determined in a cross-validation study. Compared IGRA, the ESAT-6 free IGRA induced a comparable magnitude of IFN-γ release, and the diagnostic performance was on par with Quantiferon (sensitivity 84% vs 79%; specificity 99% vs 97%). The comparable performance of the ESAT-6 free IGRA to IGRA suggests potential as companion diagnostic for ESAT-6 containing vaccines and as adjunct test for latent infection

Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6.

There is a need for an improved vaccine for tuberculosis. ESAT-6 is a cardinal vaccine antigen with unique properties and is included in several vaccine candidates in development. ESAT-6 is also the core antigen in the IFN-γ release assays (IGRA) used to diagnose latent infection, rendering IGRA tests unspecific after vaccination. This challenge has prompted the development of a companion diagnostic for ESAT-6 based vaccines, an ESAT-6 free IGRA. We screened a panel of seven potential new diagnostic antigens not recognized in BCG vaccinated individuals. Three highly recognized antigens EspC, EspF and Rv2348c were identified and combined with CFP10 in an ESAT-6 free antigen cocktail. The cocktail was prepared in a field-friendly format, lyophilized with heparin in ready-to-use vacutainer tubes. The diagnostic performance of the ESAT-6 free IGRA was determined in a cross-validation study. Compared IGRA, the ESAT-6 free IGRA induced a comparable magnitude of IFN-γ release, and the diagnostic performance was on par with Quantiferon (sensitivity 84% vs 79%; specificity 99% vs 97%). The comparable performance of the ESAT-6 free IGRA to IGRA suggests potential as companion diagnostic for ESAT-6 containing vaccines and as adjunct test for latent infection

Diagnostic benefits of adding EspC, EspF and Rv2348-B to the QuantiFERON Gold In-tube antigen combination

Interferon (IFN)-γ release assays (IGRAs) are used to diagnose latent tuberculosis (TB) infection (LTBI). To improve the accuracy of these tests, different approaches, such as alternative cytokine detection and using different antigens, are considered. Following this purpose, this study aims to evaluate the addition of EspC, EspF and Rv2348-B to those present in the QuantiFERON-TB Gold In-Tube (QFN-G-IT). We included 115 subjects: 74 active TB patients, 17 LTBI individuals and 24 healthy controls. Whole blood samples were collected in QFN-G-IT and in-house tubes containing different combinations of EspC, EspF and Rv2348-B, together with ESAT-6, CFP-10, and TB7.7. After overnight incubation at 37 ºC, plasma was harvested and IFN-γ quantified. IFN-γ levels in the QFN-G-IT and in-house tubes correlated very good (Spearman Rho(r) > 0.86). In-house antigen combinations distinguished healthy individuals from those with active TB and LTBI (specificities and sensitivities higher than 87.5% and 96.3%, respectively [AUC > 0.938]). Adding EspC, EspF and Rv2348-B, increased the sensitivity of the test, being the addition of EspC and Rv2348-B the combination that yielded a higher sensitivity with no specificity loss. Addition of these antigens could improve diagnosis in patients with impaired or immature immune response who are at high risk of developing T