| Research backgroundHepatitis B virus(HBV)infection is the leading risk factor of the development of chronic hepatitis into cirrhosis,liver failure and hepatocellular carcinoma.Hepatitis B-related hepatocellular carcinoma is the second largest cause of cancer-related death in the world[1-2].Approximately 15%-40%of untreated patients with chronic HBV infection progress to complications such as liver cirrhosis and hepatocellular carcinoma.Antiviral therapy can effectively reduce the occurrence and progress of HBV related liver diseases[3-4].Therefore,evidence-based medicine has demonstrated that effective antiviral treatment for chronic hepatitis B patients is a critical strategy to prevent serious complications.Hepatitis B surface antigen(HBsAg)clearance(with or without seroconversion to anti-HBs),undetectable DNA levels over a prolonged time,hepatitis B e antigen(HBeAg)become negative,liver biochemical parameters were normal and improvements in the histopathological examination are considered as the ideal endpoint of antiviral therapy in patients with chronic hepatitis B(CHB),and is believed to CHB clinical cure,also known as functional cure.However,patients with CHB who undergoing nucleos(t)ide analogues(NUCs)therapy HBsAg clearance rates is only 2.11%at 10 years,making it difficult to achieve a functional cure[5].Seroconversion of HBeAg is an important basis and prerequisite for clinical cure.HBeAg positive CHB patients can obtain sustained virologic response,ALT normalized and HBeAg seroconversion;HBeAg-negative CHB patients can receive sustained virologic response and ALT normalized will make it easier to achieve the"ideal endpoint".Therefore,for the CHB patients with NUCs treated and persistently undetectable HBV DNA to below the limit of detection for a long period of time,further screening the dominant population of HBsAg clearance and developing appropriate treatment strategies may greatly improve the HBsAg clearance rate.The main antiviral drugs available are divided into two categories,including direct antivirals and immunomodulators.Single antiviral drug appeared to have a limited effect on CHB clinical cure[6-7].Recent studies have shown that NUCs sequential or combined with Pegylated interferon(Peg-IFN)therapy may increase the HBsAg clearance rate in CHB patients with NUCs treated[8-9].Theoretically speaking,in CHB patients undergoing long-lasting NUCs treatment,HBV DNA replication is fully inhibited,which contributes to the recovery of HBV-specific CD8+T cells,and further combination with interferon can promote the decrease of viral antigens in the liver.Interferon can enhance NK cell activity,activate T lymphocyte immune response,inhibit the formation of HBV protein,increase hepatocellular injury and compensatory hepatocyte proliferation,and promote the instability of cccDNA in hepatocytes[10-11].OSST studies[12]and New Switch studies[31]have shown that HBeAg negative patients with serum HBsAg levels<1500 IU/mL and HBV DNA is lower than the lowest detection limit,after 48 weeks of sequential Peg-IFN therapy,HBsAg clearance rates are 22.0%to 25.4%.HBsAg levels<1500 IU/ml is an important index for predicting HBsAg clearance after interferon treatment.Therefore,we speculate that the combination therapy with NUCs plus Peg-IFNα in CHB patients with low levels of HBsAg can promote HBsAg loss.At the same time,it can reduce risk of HBV DNA reactivation caused by discontinuation of NUCs during sequential therapy.This may be a more optimized strategy in the current combination therapy.However,there is insufficient evidence of superiority in patient was advised to undergo NUCs plus Peg-IFN combination therapy,and there is no uniform standard for treatment population,combination therapy and treatment time.Chronic hepatitis B patients with low levels of HBsAg,HBeAg-negative,HBV DNA to below the limit of detection and receiving a stable NUCs treatment were enrolled in this prospective study.Enrolled patients were treated with Peg-IFNa to explore the efficacy of combined treatment for clinical cure.The baseline factors and the changes of HBsAg kinetics during treatment were analyzed to predict serum HBsAg clearance in CHB patients.This is helpful to improve the clinical recovery rate of CHB patients receiving NUCs.ObjectiveTo investigate the clearance of serum HBsAg by addition of Peg-IFNa to a long-lasting NUCs therapy in a HBeAg negative,HBV DNA negative(<500 IU/mL),low levels of HBsAg(≤ 1500 IU/mL)chronic hepatitis B patient.To observed the dynamic evolution of serum HBsAg during combination treatment,the relationship between serum HBsAg level and decreases at each follow-up time and HBsAg seroconversion are explored,so as to provide a reliable basis for the optimization of clinical cure for hepatitis B.Methods1.Chronic hepatitis B patients with HBsAg ≤1500 IU/mL,HBeAg negative,persistently undetectable HBV DNA to below the limit of detection(<500 IU/mL)for a long period of time and receiving a stable NUCs treatment for at least 1 year were enrolled in this prospective study during 2018-2019 at the Hospital according to the diagnostic criteria of "Guideline of Prevention and Treatment for Chronic Hepatitis B(2015 Update)"[14].Patients were excluded if they had cirrhosis,liver decompensation,hepatocellular carcinoma and co-infection with other types of hepatitis virus and HIV.Enrolled patients were treated with a 48-week add-on course of Peg-IFNα,if HBsAg decline titer is more than 1 log10 or the absolute value is less than 100 IU/mL,extension the treatment of Peg-IFN to 72 weeks may be considered for CHB patients,but the total course of treatment is not more than 96 weeks.The main end point of the study was the proportion of HBsAg clearance at 72 weeks,and the predictors of HBsAg clearance were analyzed.2.The symptoms,signs,blood routine,blood biochemistry and electrolytes,HBV DNA quantification,HBsAg,HBeAg,HBsAb quantitative detection,alpha-fetoprotein(AFP),liver ultrasonography(US)and liver transient elastography,and so on were monitored at each follow-up point during the combined treatment period.There were 10 follow-up points in this study,including screening period,baseline period,treatment period at 4 weeks,8 weeks,12 weeks,24 weeks,36 weeks,48 weeks,60 weeks and 72 weeks.3.The statistical data were performed using SPSS23.0 statistical software.Continuous variables are expressed as mean ± standard deviation(normally distributed continuous variables)or median and interquartile range(non-normally distributed continuous variables),and categorical variables are presented as percentage.Quantitative data were tested using T-test or non-parametric test,qualitative data were compared by Fisher exact test.Repeated measures analysis of variance is used to compare the changes of HBsAg level and decreasing amplitude among groups at different time points.The proportion of patients with serum HBsAg clearance after combined treatment in patients with different baseline HBsAg levels was estimated using Kaplan-Meier method,comparison between groups were done by log rank test.Univariate and multivariate logistic regression analysis and receiver operating characteristic curve(ROC)were used to determine the predictive factors of HBsAg clearance.HBsAg quantitative was log10 transformed to adjust the non-normality of the original data distribution.Differences were was considered to be statistically significant when P<0.05.Results1.A total of 38 patients were included in the current study,13 patients achieved HBsAg clearance after 48 weeks of combined treatment,and 6 patients obtained HBsAg clearance during 72 weeks of treatment,accounting for 50.00%of all enrolled patients.2.Enrolled patients were classified into clearance group and non-clearance group based on whether HBsAg loss at the end of treatment.The difference of baseline characteristics between clearance group and non-clearance group was observed,and the results showed that age and baseline HBsAg level had statistically significant between two groups(P<0.05).There was no significant difference in Gender,baseline ALT level and other factors between the two groups.3.After receiving NUCs plus Peg-IFNα combination therapy for 24 weeks,48 weeks and 72 weeks,HBsAg clearance rates were 21.05%(8/38),34.21%(13/38)and 50.00%(19/38),respectively,while HBsAg seroconversion rates were 10.54%(4/38),23.68%(9/38)and 42.11%(16/38),respectively.The results showed that HBsAg level after combination therapy for 72 weeks was significantly lower than baseline HBsAg level(P<0.001).Combined therapy with Peg-IFNα could substantially reduce serum HBsAg level.4.Patients were further divided into three groups(baseline HBsAg levels<50 IU/mL,50-1000 IU/mL and 1000-1500 IU/mL).Patients with baseline HBsAg levels<1000 IU/mL at week 72 had higher HBsAg loss rates after therapy than those with baseline HBsAg levels≥1000 IU/mL(75.0%vs 0.0%;52.0%vs 0.0%;P<0.05).And HBsAg clearance rate increased with decreasing baseline HBsAg levels(P<0.05).5.The HBsAg levels in CHB patients showed a continuous declined trend during NUCs plus Peg-IFNα combination treatment,especially declines were greatest after combination therapy for 24 weeks(P<0.05).There was a significant difference in serum HBsAg kinetics between HBsAg clearance group and non-clearance group(P<0.05),and patients in the clearance group showed a significantly larger decrease in HBsAg levels than non-clearance group after treatment for 12 weeks and 24 weeks(P=0.007).Patients were divided into three groups(baseline HBsAg levels<50 IU/mL,50-1000 IU/mL and 1000-1500 IU/mL).There was significant difference in serum HBsAg kinetics among the three groups during combined treatment(P<0.05).6.Univariate logistic regression analysis showed that age,HBsAg levels at baseline,12 and 24 weeks,HBsAg levels reduction in early treatment were predictors of HBsAg clearance at 72 weeks.Multivariate logistic regression analysis showed that age[odds ratio(OR)=1.311;P=0.016;95%confidence interval(CI):1.051-1.635]and HBsAg levels at weeks 24(OR=4.481;P=0.004;95%CI:1.634-12.290)were an independent predictor of HBsAg loss.7.Multiple factors were observed to predict HBsAg clearance,patients with aged≤38 years and HBsAg levels ≤0.92 log10IU/mL at weeks 24 had an HBsAg clearance rate of 100.0%(12/12);patients with aged≤38,baseline HBsAg levels ≤2.86 log10IU/mL,HBsAg levels ≤0.92 log10IU/mL at weeks 24 and weeks 12 HBsAg change from baseline≥0.67 log10IU/mL had an HBsAg clearance rate of 100.0%(9/9),while HBsAg clearance rate was 0.0%(0/6).8.The safety of the combination therapy with NUCs plus Peg-IFN-α in CHB patients was satisfactory,and there were no treatment-related life-threatening serious adverse events or deaths related to the treatment.ConclusionThe HBeAg negative chronic hepatitis B patients with receiving a stable NUCs treatment,HBsAg levels≤1500 IU/mL and HBV DNA is undetectable were treated with Peg-IFNa can promote HBsAg clearance.Among the 16 patients who did not achieve HBsAg clearance after combination therapy for 48 weeks,6 patients(37.50%)obtained HBsAg clearance after extending the course of treatment to 72 weeks.Therefore,extend the cycle of treatment and the best individualized therapies strategy tailored on the basis of predictors,rather than a fixed course of 48 weeks.We have demonstrated the age(≤ 38),baseline HBsAg levels(≤2.86 log10IU/mL),HBsAg levels at weeks 24(≤ 0.92 log10IU/mL)and weeks 12 HBsAg levels decline from baseline(≥ 0.76 log10IU/mL)can be used as important indicator to predict serum HBsAg clearance after combination therapy.The combined indicator underlying HBsAg levels ≤0.92 log10IU/mL at weeks 24 will identify 85.0%-100.0%of the patients who are likely to obtain HBsAg clearance after 72 weeks of combined treatment.In addition,age was associated with HBsAg clearance(OR=1.311;P=0.016),which means that younger patients will have more opportunity to achieve HBsAg 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