| BackgroundHepatitis B virus(HBV)is one of the most common chronic viral infection in the world with approximately 350 million infected patients,leading to the development of chronic liver injury,cirrhosis and hepatocellular carcinoma.Nucleos(t)ide analogues(NAs)are widely used in clinical practice because of their efficient inhibitory in virus replication,portability and safety.However,NAs cannot eliminate intrahepatic cccDNA,and few patients can achieve HBsAg clearance during treatment,the so-called clinical cure.So far,there is no consensus on the best time to stop NAs therapy recommended by the guidelines.For most patients with chronic hepatitis B(CHB),they require long-term or even life-long treatment of NAs.Therefore,they may face the problems including psychological and economic burden,medication side-effects,as well as poor compliance with treatment.Obviously,an unfinite long-term treatment is not an ideal selection.However,the sensitive and specific predictive biomarkers to guide the discontinuation of NA therapy is an unmet need.We aim to investigate the serum HBcrAg levels and T cell receptor(TCR)immune repertoire of patients in a prospective cohort who stopped NAs according to the guideline,and analyzed their association with clinical relapse.MethodWe conducted a prospective,open-label cohort study from November,2011.The patients who met the inclusion and exclusion criteria and given full informed consent,were enrolled in this study.Inclusion criteria:HBeAg-positive non-cirrhotic patients were treated with oral Nucleos(t)ide Analogues,and achieved a complete response(defined as HBV DNA was lower than the detection line,ALT level returned to normal,and HBeAg seroconversion occurred),another more than 1 year consolidation therapy was also needed.The endpoint of the study is clinical relapse,defined as HBV DNA>2000IU/mL at any time point during the follow-up,accompanied by ALT≥2ULN,and ALT is still ≥2ULN after re-examination within 2 weeks,while liver protection drugs are not allowed during this period.Additionally,other causes of abnormal ALT levels should be excluded.Result1.Combining serum HBcrAg and HBsAg can predict clinical relapse after NA discontinuation:A total of 78/122(63.9%)patients experienced sustained response after NAs cessation,and 44/122(36.1%)patients experienced clinical relapse.In multivariate analysis,EOT HBcrAg(hazard ratio[HR]=2.105 95%CI:1.440-3.077,p<0.001),hepatitis B surface antigen(HBsAg)≥100 IU/mL(HR=4.406,95%CI 1.567-12.389,p=0.005)and age(HR=1.051,95%CI:1.0101.093,p=0.049)were independently associated with clinical relapse.A cut-off value of 4.0 log10 U/mL of HBcrAg was defined by maximized Youden’s index.An EOT HBcrAg level of≥4.0 log10 U/mL was associated with higher risks of clinical relapse(65.8%vs.23.2%,p<0.001)and HBeAg reversion(27.5%vs.1.6%,p<0.001).In majority of patients(n=91)who had a high EOT HBsAg level(≥100 IU/mL),serum HBcrAg level could further discriminate patients at low risk of clinical relapse.Patients with an HBcrAg level≥4.0 log10 U/mL had significantly higher cumulative incidence rates of clinical relapse(78.1%vs.29.4%,p<0.001)and HBeAg reversion(29.4%vs.0%,p<0.001).To explore the TCR repertoires in patients with NA discontinuaiton,19 patients with positive HBeAg before treatment and HBsAg<3500 IU/mL before NA discontinuation were included(10 patients were sustained response within 96 weeks(NF group)and 9 patients with clinical relapse(F group)).The T1 time point was the baseline of NA discontinuation;the T2 time point was 8 weeks or 12 weeks,and the T3 time point was 24 weeks or the clinical relapse time point.2.1 The CDR3 clonal diversity of CD4 cells in the F group and the NF group was different at the baseline of NA discontinuation.The CDR3 clonal diversity of CD4 cells in group F increased at time points T1 to T2 and time points T1 to T3.At the T3 time point,the VDJ combination of CD4 and CD8 cells was significantly higher in the NF group than in the F group.The increase of TCR CDR3 library diversity was positively correlated with the increase of HBV DNA level(r=-0.630,p=0.001).2.2 There were obvious new clones of CD4 cells CDR3 in group F patients after NA discontinuation.From T2 to T3 time points,the proportion of CDR3 newborn clones in CD4 cells was positively correlated with ALT and HBV DNA levels and their changes,and tended to be positively correlated with HBsAg and their changes.2.3 Patients in group F had higher frequency of high-frequency CDR3 clones and high-frequency persistence index of CD4 cells.The persistence index of the top 100 high-frequency clones(that is,the percentage of CDR3 clones that persisted from the previous time point to the next time point)was significantly higher in the top 10～21 clonal persistence index of CD4 cells in group F at any time point.in the NF group.And from T2 to T3,there was a positive correlation between TCPI and ALT/HBsAg/HBV DNA levels.2.4 The highest frequency of CD8 CDR3 clones can discriminate patients with different clinical outcomes in a systematic analysis.The CDR3 amino acid sequences of 8/9 patients in the F group continued to be top 1 clones and 9/10 patients of the NF group continued to have top 1 clones.The CDR3 amino acid sequences formed two distinct parts on the evolutionary tree,suggesting that the CDR3 amino acid sequences of the CD8 continuous top 1 clones were in the F group.and NF group were significantly different.The phylogeny analysis of the CDR3 sequences of the top1 CD4 clone was also performed,and it was found that the F group and the NF group could not be well differentiated.2.5 The overlapping rate of CD4 cells CDR3 clones in patients in the sustained response group was higher.The similarity of the TRB CDR3 library between the two patients within the group showed that in the CD4 cell subset,the similarity of the TRB CDR3 library between individuals in the NF group was significantly higher than that in the F group patients,and in the CD8 cell subset within the two groups There was no statistical difference in the similarity of TRB CDR3 libraries between individuals.Conclusion:1.The novel viral biomarker HBcrAg is associated with clinical relapse after NA discontinuation,which is a potential marker to guide a safe finite NA treatment.2.TCR sequencing could find the differences of CDR3 clones of T cells in patients with different clinical outcomes after NA discontinuation.The observation of the characteristics and dynamic changes of CDR3 clones in patients,may be helpful for physicians to understand the immune changes of T cells in patients after NA discontinuation. |
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