Routine evaluation of HBV-specific T cell reactivity in chronic hepatitis B using a broad-spectrum T-cell epitope peptide library and ELISpot assay

Abstract

Abstract Background The clinical routine test of HBV-specific T cell reactivity is still limited due to the high polymorphisms of human leukocyte antigens (HLA) in patient cohort and the lack of universal detection kit, thus the clinical implication remains disputed. Methods A broad-spectrum peptide library, which consists of 103 functionally validated CD8+ T-cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and fits to the HLA polymorphisms of Chinese and Northeast Asian populations, was grouped into eight peptide pools and was used to establish an ELISpot assay for enumerating the reactive HBV-specific T cells in PBMCs. Totally 294 HBV-infected patients including 203 ones with chronic hepatitis B (CHB), 13 ones in acute resolved stage (R), 52 ones with liver cirrhosis (LC) and 26 ones with hepatocellular carcinoma (HCC) were detected, and 33 CHB patients were longitudinally monitored for 3 times with an interval of 3–5 months. Results The numbers of reactive HBV-specific T cells were significantly correlated with ALT level, HBsAg level, and disease stage (R, CHB, LC and HCC), and R patients displayed the strongest HBV-specific T cell reactivity while CHB patients showed the weakest one. For 203 CHB patients, the numbers of reactive HBV-specific T cells presented a significantly declined trend when the serum viral DNA load, HBsAg, HBeAg or ALT level gradually increased, but only a very low negative correlation coefficient was defined (r = − 0.21, − 0.21, − 0.27, − 0.079, respectively). Different Nucleotide Analogs (NUCs) did not bring difference on HBV-specific T cell reactivity in the same duration of treatment. NUCs/pegIFN-α combination led to much more reactive HBV-specific T cells than NUCs monotherapy. The dynamic numbers of reactive HBV-specific T cells were obviously increasing in most CHB patients undergoing routine treatment, and the longitudinal trend possess a high predictive power for the hepatitis progression 6 or 12 months later. Conclusion The presented method could be developed into an efficient reference method for the clinical evaluation of cellular immunity. The CHB patients presenting low reactivity of HBV-specific T cells have a worse prognosis for hepatitis progression and should be treated using pegIFN-α to improve host T-cell immunity.

Keywords

• Chronic hepatitis B
• Antigen-specific T cell
• T-cell epitopes
• ELISpot