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

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: Antigen-specific T cell; Chronic hepatitis B; ELISpot; T-cell epitopes.

Fig. 1

HBV-specific T cell reactivity in 294 HBV-infected patients at different disease stages. Reactive HBV-specific T cells in PBMCs were detected using ex vivo IFN-γ ELISpot assay and 103 validated T-cell epitope peptides. A Total HBV-specific T cells (SFUs) in HBV-infected patients at different disease stages (R, n = 13; CHB, n = 203; LC, n = 52; HCC, n = 26). B Deconvolution of HBV-specific T cells from total antigens into the indicated HBV protein (HBsAg, HBpol, HBx, HBeAg) in HBV-infected patients. C Total HBV-specific T cells (SFUs) in CHB patients at different clinical phases (IA, n = 23; IT, n = 24; IC, n = 44). D Deconvolution of HBV-specific T cells from total antigens into the indicated HBV protein (HBsAg, HBpol, HBx, HBeAg) in CHB patients. Medians (interquartile range) were presented and statistical analyses were performed using Kruskal–Wallis test (K–W) across multiple groups and Mann–Whitney test (M–W) between two groups

Fig. 2

Association of HBV-specific T cell reactivity with sero-virological parameters in CHB patients. A Stratified analyses of HBV-specific T cells (SFUs) in CHB patients grouped by HBV DNA load (< 3.0, n = 70; 3.0–5.0, n = 27; > 5.0, n = 23), HBsAg level (< 1000, n = 82; 1000–10000, n = 64; > 10,000, n = 34), HBeAg level (< 1, n = 53; 1–100, n = 45; 100–1000, n = 13; > 1000, n = 15) and ALT level (< 40, n = 146; > 40, n = 56). B Stratified analyses of sero-virological parameters in CHB patients grouped by HBV-specific T cell reactivity (0–24 SFUs for 25% of the cohort; 25–90 SFUs for 50% of the cohort; 91–622 SFUs for 25% of the cohort). For HBV DNA load, HBsAg level, HBeAg level, and ALT level analyses, 0–24 group, n = 42, 27, 41, 50, respectively; 25–90 group, n = 93, 59, 77,101, respectively; 91–622 group, n = 45, 24, 52, 51, respectively. C Spearman correlation tests between HBV-specific T cells (SFUs) and HBV DNA, HBsAg, HBeAg or ALT levels

Fig. 3

Association of HBV-specific T cell reactivity with anti-virus therapy in CHB patients. A HBV-specific T cells (SFUs) in CHB patients with different treatments. B Specific T cells (SFUs) reactive to each HBV protein (HBsAg, HBpol, HBx, HBeAg) in different treatment groups. Untreated group, n = 15; NUCs monotherapy, n = 167; NUCs/IFN combination therapy, n = 21. C HBV-specific T cells (SFUs) after NUCs treatment in different durations of treatment. D HBV-specific T cells (SFUs) after different NUCs treatment in the same treatment duration. Medians (interquartile range) were presented and statistical analyses were performed using Kruskal–Wallis test (K–W) across multiple groups and Mann–Whitney test (M–W) between two groups

Fig. 4

Dynamic changes of HBV-specific T cells and sero-virological parameters in CHB patients. 33 CHB patients undergoing routine treatment were followed by HBV-specific T cell detection and sero-virological parameters collections for three times at an interval of 3–5 months. A, B Dynamic changes of total HBV-specific T cells and the specific T cells reactive to each HBV protein in 33 CHB patients. C Dynamic changes of HBV DNA (n = 18), HBsAg (n = 27), HBeAg (n = 19), ALT (n = 32), and AST (n = 32) levels. Then, the dynamic changes of HBV-specific T cells in CHB patients with different fluctuation courses of D HBV DNA load (decrease, n = 7; no alternation, n = 3; increase, n = 2), E HBsAg level (decrease, n = 18; no alternation, n = 10), F HBeAg level (seroconversion, n = 4; retained, n = 13), and G ALT level (normal, n = 14; decrease, n = 12; increase, n = 7) were presented. The patients who achieved DNA fluctuations (increase or decrease) > 30% were defined as the DNA-increase or DNA-decrease group, and the other patients were defined as DNA-no alternations group. HBsAg-decrease was defined as an amplitude decrement of more than 30%. CHB patients who experienced a positive HBeAg serology (HBeAg COI > 1) at first and seroconverted (HBeAg COI < 1) later were defined as the HBeAg-seroconversion group. ALT-decrease was defined as a decline to the normal range (< 40 IU/L) or decreased more than 30%, while ALT that rose more than 30% or beyond 40 IU/L was defined as ALT-increase. The paired, two-tailed Student’s t tests between two groups and Kruskal–Wallis test (K–W) across more than two groups were performed

Fig. 5

Dynamic changes of sero-virological parameters during different fluctuation courses of HBV-specific T cells. 33 CHB patients were followed by HBV-specific T cell detection and sero-virological parameters collections for three times at an interval of 3–5 months. According to the dynamic courses of HBV-specific T cells (SFUs), patients were categorized as ascending (A), ascending/descending (B), stationary, descending, or descending/ascending groups, then HBV DNA load, HBsAg, HBeAg, HBx, HBpol and ALT levels were longitudinally analyzed. The SFU numbers of reactive HBV-specific T cells which increased or decreased more than 50% than last test were defined as ascending or descending during the follow-up period

Fig. 6

Predictive power of cross-sectional reactivity of HBV-specific T cells for hepatitis progression in CHB patients. CHB patients were divided into normal (ALT < 40 IU/L) group and abnormal (ALT > 40 IU/L) group of liver function at the test time of HBV-specific T cells or 6 months later after the test. ROC curve analyses of DNA load (IU/mL), HBV-specific T cells (SFUs/2 × 10⁵ PBMCs), and a combination were performed to predict hepatitis progression at the test time of HBV-specific T cells (A) and 6 months later after the test (B), using R package pROC, and summarized in table. ROC, receiver operating characteristic; AUC, area under the curve; PPV, positive predictive value; NPV, negative predictive value. The p¹ values represent the significance of model. The p² values represent the significance of difference between the AUC of combined markers (DNA load + Specific T cell) and single predictor

Fig. 7

Predictive power of longitudinal reactivity of HBV-specific T cells for liver hepatitis progression in CHB patients. 33 CHB patients undergoing NUCs or NUCs/INF-α treatment were divided into normal (ALT < 40 IU/L) group and abnormal (ALT > 40 IU/L) group of liver function at 6 months (normal/abnormal: 8/25) or 12 months (normal/abnormal: 6/27) after the last test of HBV-specific T cells. ROC curve analyses of single test and combined tests of HBV-specific T cells (SFUs/2 × 10⁵ PBMCs) were performed to predict hepatitis progression at 6 months (A) and 12 months after the last test (B) of HBV-specific T cells, using R package pROC, and summarized in table. The p¹ values represent the significance of model. The p² values represent the significance of difference between the AUC of combined markers (First + Second + Third) and other predictors