title: Development of a novel IGRA assay to test T cell responsiveness to HBV antigens in whole blood of chronic Hepatitis B patients.
abstract: 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 IFNγ- 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 naïve and treated CHB patients showed a weaker IFNγ 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 IFNγ 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 IFNγ 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 %.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg, 20246, Germany. w.dammermann@uke.de.
authors.forename: Werner
authors.initials: W
authors.lastname: Dammermann
authors.affiliation: Department of Transfusion Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg, 20246, Germany. f.bentzien@uke.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Bentzien
authors.affiliation: Department of Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg, 20246, Germany. eva-maria.stiel@nucch.de.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Stiel
authors.affiliation: Department of Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg, 20246, Germany. kuehne-c@gmx.de.
authors.forename: Claudia
authors.initials: C
authors.lastname: Kühne
authors.affiliation: Department of Anatomy and Experimental Morphology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. s.ullrich@uke.de.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Ullrich
authors.affiliation: Department of Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg, 20246, Germany. j.schulze-zur-wiesch@uke.de.|German Center for Infection Research (DZIF), partner site Hamburg, Hamburg, Germany. j.schulze-zur-wiesch@uke.de.|Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany. j.schulze-zur-wiesch@uke.de.
authors.forename: Julian
authors.initials: J
authors.lastname: Schulze Zur Wiesch
authors.affiliation: Department of Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg, 20246, Germany. s.lueth@gmx.de.|German Center for Infection Research (DZIF), partner site Hamburg, Hamburg, Germany. s.lueth@gmx.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Lüth
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D005260:Female; D006512:Hepatitis B Core Antigens / Q000097:blood*; D006514:Hepatitis B Surface Antigens / Q000097:blood*; D006515:Hepatitis B virus / Q000187:drug effects / Q000276:immunology*; D019694:Hepatitis B, Chronic / Q000097:blood* / Q000188:drug therapy / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000097:blood; D059425:Interferon-gamma Release Tests / Q000379:methods*; D007376:Interleukin-2 / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009711:Nucleotides / Q000494:pharmacology / Q000627:therapeutic use; D010455:Peptides / Q000276:immunology; D012372:ROC Curve; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D014611:Vaccination
chemical_list: D006512:Hepatitis B Core Antigens; D006514:Hepatitis B Surface Antigens; D007376:Interleukin-2; D009711:Nucleotides; D010455:Peptides; D007371:Interferon-gamma