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

Dammermann W, Bentzien F, Stiel EM, Kühne C, Ullrich S, Schulze Zur Wiesch J, Lüth S - J Transl Med (2015)

Bottom Line: 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).For HC, IL2 release after HBsAg stimulation depicted hepatitis B vaccination status with a diagnostic sensitivity and specificity of 85 % and 90 %.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg, 20246, Germany. w.dammermann@uke.de.

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 %.

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.

No MeSH data available.


Related in: MedlinePlus

IL2 responses to HBsAg in HC correlate with hepatitis B vaccination status. (a) Receiver-operating-characteristic (ROC) curve. AUC, area under the curve. (b) Scatter plot of IL2 release of HepB vaccinated and not vaccinated HC after HBsAg stimulation of whole blood. Negative control values were deducted from the antigen induced responses. Dotted line indicates IL2 cut-off at 11 pg/ml. n = 74 HC, whereas n = 33 were anti-HBs positive and n = 41 anti-HBs negative
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Fig5: IL2 responses to HBsAg in HC correlate with hepatitis B vaccination status. (a) Receiver-operating-characteristic (ROC) curve. AUC, area under the curve. (b) Scatter plot of IL2 release of HepB vaccinated and not vaccinated HC after HBsAg stimulation of whole blood. Negative control values were deducted from the antigen induced responses. Dotted line indicates IL2 cut-off at 11 pg/ml. n = 74 HC, whereas n = 33 were anti-HBs positive and n = 41 anti-HBs negative

Mentions: HBsAg constitutes the single main antigen in hepatitis B vaccine formulations. Thus, 74 HC with known hepatitis B vaccination status were also analyzed regarding their HBsAg-dependent IFNγ and IL2 release. 33/74 HC possessed a positive anti-HBs antibody titer and 41/74 were anti-HBs negative. Whereas IFNγ responses were negligible, IL2 values reached marked heights if the tested individual had been successfully vaccinated (Fig. 5b), but these IL2 levels did not correlate with the corresponding anti-HBs antibody titer (Fig. 6). To determine the cut-off value of our newly developed assay, we performed a ROC analysis of the IL2 readings (Fig. 5a). This HBV specific cytokine release assay with HBsAg-specific peptides using IL2 release as the primary readout (cut-off = 11 pg/ml IL2) reached 85 % diagnostic sensitivity and 90 % diagnostic specificity. The corresponding AUC value was at 0.92. Using the cut-off defined by the ROC analysis, as shown in Fig. 4, 28 (of 33) vaccinated HC showed positive scores, while 4 (of 41) not vaccinated HC slightly exceeded the cut-off value (Fig. 5b).Fig. 5


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

Dammermann W, Bentzien F, Stiel EM, Kühne C, Ullrich S, Schulze Zur Wiesch J, Lüth S - J Transl Med (2015)

IL2 responses to HBsAg in HC correlate with hepatitis B vaccination status. (a) Receiver-operating-characteristic (ROC) curve. AUC, area under the curve. (b) Scatter plot of IL2 release of HepB vaccinated and not vaccinated HC after HBsAg stimulation of whole blood. Negative control values were deducted from the antigen induced responses. Dotted line indicates IL2 cut-off at 11 pg/ml. n = 74 HC, whereas n = 33 were anti-HBs positive and n = 41 anti-HBs negative
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4465460&req=5

Fig5: IL2 responses to HBsAg in HC correlate with hepatitis B vaccination status. (a) Receiver-operating-characteristic (ROC) curve. AUC, area under the curve. (b) Scatter plot of IL2 release of HepB vaccinated and not vaccinated HC after HBsAg stimulation of whole blood. Negative control values were deducted from the antigen induced responses. Dotted line indicates IL2 cut-off at 11 pg/ml. n = 74 HC, whereas n = 33 were anti-HBs positive and n = 41 anti-HBs negative
Mentions: HBsAg constitutes the single main antigen in hepatitis B vaccine formulations. Thus, 74 HC with known hepatitis B vaccination status were also analyzed regarding their HBsAg-dependent IFNγ and IL2 release. 33/74 HC possessed a positive anti-HBs antibody titer and 41/74 were anti-HBs negative. Whereas IFNγ responses were negligible, IL2 values reached marked heights if the tested individual had been successfully vaccinated (Fig. 5b), but these IL2 levels did not correlate with the corresponding anti-HBs antibody titer (Fig. 6). To determine the cut-off value of our newly developed assay, we performed a ROC analysis of the IL2 readings (Fig. 5a). This HBV specific cytokine release assay with HBsAg-specific peptides using IL2 release as the primary readout (cut-off = 11 pg/ml IL2) reached 85 % diagnostic sensitivity and 90 % diagnostic specificity. The corresponding AUC value was at 0.92. Using the cut-off defined by the ROC analysis, as shown in Fig. 4, 28 (of 33) vaccinated HC showed positive scores, while 4 (of 41) not vaccinated HC slightly exceeded the cut-off value (Fig. 5b).Fig. 5

Bottom Line: 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).For HC, IL2 release after HBsAg stimulation depicted hepatitis B vaccination status with a diagnostic sensitivity and specificity of 85 % and 90 %.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg, 20246, Germany. w.dammermann@uke.de.

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 %.

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.

No MeSH data available.


Related in: MedlinePlus