Development of a novel IGRA assay to test T cell responsiveness to HBV antigens in whole blood of chronic Hepatitis B patients

Background: Interferon gamma release assays (IGRA) have been developed to support easy and fast diagnosis of diseases like tuberculosis, and CMV in transplant patients. IGRAs focus on cellular immunity especially memory T cells and thus also allow rapid screening prior to complex flow cytometric testing. Here, we describe a novel, sensitive whole blood based cytokine release assay capable of assessing T cell responsiveness to HBV antigens in Hepatitis B patients and assessing hepatitis B vaccination status in healthy individuals. Methods: Seventy two chronic Hepatitis B patients (CHB), 8 acute hepatitis B patients (AHB) and 80 healthy controls (HC) were tested by ELISA for IFN gamma- and IL2-secretion in whole blood after challenge with synthetic peptide libraries of hepatitis B core antigen (HBcAg) or hepatitis B surface antigen (HBsAg). Results: The developed IGRA test reliably differentiated between Hepatitis B patients, vaccinees and unvaccinated healthy controls. Treatment naive and treated CHB patients showed a weaker IFN gamma response to HBcAg (16 +/- 5 and 35 +/- 28 pg/ml, respectively) compared to the AHB group (82 +/- 39 pg/ml), whereas HC remained unresponsive (6 +/- 1 pg/ml). IL2 levels after HBcAg challenge were also higher in the AHB group compared to naive and treated CHB as well as HC (47 +/- 21 vs. 12 +/- 3, 15 +/- 10 and 12 +/- 9 pg/ml, respectively). HBsAg stimulation led to increased IFN gamma and IL2 levels in the AHB group (33 +/- 12 and 22 +/- 12 pg/ml) and even higher levels in HC due to a high hepatitis B vaccination rate (41 +/- 10 and 167 +/- 58 pg/ml). Naive and treated CHB patients developed no or only weaker IFN gamma or IL2 responses to HBsAg (5 +/- 2 and 12 +/- 7 pg/ml, for naive CHB, 12 +/- 10 and 18 +/- 15 pg/ml, for treated CHB). For HC, IL2 release after HBsAg stimulation depicted hepatitis B vaccination status with a diagnostic sensitivity and specificity of 85 % and 90 %. Conclusion: Our novel whole blood based cytokine release assay constitutes an easy and robust tool for screening HBV specific cellular immunity as alternative to flow cytometry or ELISPOT assays.