A New Method To Improve The Functional Cure Rate Of Chronic Hepatitis B And Preliminary Study Of Immunologic Mechanism Based On ScRNA-seq

Background Hepatitis B virus（HBV）infection is a global public health problem,which seriously threatens human health.For patients with chronic hepatitis B（CHB）,to achieve functional cure,which means the serological clearance of hepatitis B surface antigen（HBsAg）,is the ideal goal of antiviral therapy.Nowadays,the antiviral drugs for CHB mainly include interferon and nucleos（t）ide analogues,both of which can effectively inhibit HBV replication and prevent the disease progression.However,it is difficult to achieve functional cure and the rate of HBsAg loss is very low.Therefore,to explore and apply new methods to improve the functional cure rate of CHB and to find new indicators to predict the curative effect in the process of treatments has become an important research direction.Exploring the host’s immunologic changes in the process of antiviral therapy,not only helps to guide treatment,but also helps to find new drug targets.At present,single cell sequencing has become the focus of research in the field of life science.Using the single-cellRNA sequencing（scRNA-seq）technology to explore the distribution,status,action process and cooperation mechanism of different immune cell subsets before and after CHB treatment from the single cell level is expected to provide a new insight for clinical diagnosis and treatment of CHB.Objective This study was to compare and evaluate the clinical efficacy of the new antiviral/immunomodulatory combination therapy regimens in na（?）ve CHB patients and test new serological markers at different sites in the course of the finite antiviral treatment and try to analyze its dynamic changes and explore its prediction of efficacy for antiviral treatment.Besides,preliminarily exploration of the changes of immune cells in CHB patients before and after treatment with single-cellRNA sequencing（scRNA-seq）was carried out.Methods Part Ⅰ:A total of 180 HBeAg positive na（?）ve CHB patients were enrolled and randomly divided into three groups（I:Peg IFNα-2b;II:Peg IFNα-2b+TDF;III:Peg IFNα-2b+TDF+GM-CSF+HBV vaccine）for 48 weeks’antiviral treatments.The primary endpoint was the rate of HBsAg loss at the 48th week.Part Ⅱ:The serum levels of hepatitis B core-related antigen（HBcrAg）and HBV pregenomicRNA（pgRNA）of the 70 patients in the first part of the cohort were tested at week 0,4,12,24,36 and 48,respectively.And their dynamic changes in the antiviral course of CHB and the prediction related to treatment response were analyzed.Part Ⅲ:Nine patients with CHB were enrolled,4 of whom received 48 weeks of Peg IFN-α-2b+TDF.Peripheral blood mononuclear cells（PBMCs）were isolated at week0 and 48 for scRNA-seq.And combined with single cell sequencing data of healthy people,the changes of immune cell subsets in patients with CHB before and after treatment were analyzed.Results Part Ⅰ:At the week of 48,the rate of HBsAg loss in groups with Peg IFNα-2b,Peg IFNα-2b+TDF,Peg IFNα-2b+TDF+GM-CSF+hepatitis B vaccine was 0%（0/57）,3.3%（2/60）and 10.2%（6/59）,respectively.The difference among the three groups were statistically significant（χ²=6.546,P=0.021）,and the rate of HBsAg loss in Peg IFN-α-2b+TDF+GM-CSF+HBV vaccine group was higher than that of the group with Peg IFN-α-2b（P=0.027）.Part ⅡⅠ:70 patients were divided into two groups according to whether the HBsAg level was declined≥1 log₁₀ IU/m L at week 48 compared with the baseline.In the two groups,HBcrAg and pgRNA was positively correlated with HBsAg,HBeAg and HBV DNA,respectively（all r>0.5,P<0.001）.ΔpgRNA at week 24 was independently correlated withΔHBsAg≥1 log₁₀ IU/m L at week 48（P<0.001）,and the AUC was 0.754（95%CI=0.643-0.865）.Part Ⅲ:Eight immune cell subsets were captured by scRNA-seq.Compared with healthy people,pro-inflammatory CD14⁺and CD16⁺monocyte subsets were found in CHB patients.And the proportion of IFNG⁺CX3CR1^-NK cells increased significantly,which was closely correlated with the level of HBsAg（all r>0.7,P<0.019）.Besides,the expression of proinflammatory monocyte subsets decreased,the proportion of initial NK cells increased,and the T cell exhaustion was alleviated with treatment.Conclusion The new antiviral/immunomodulatory combination therapy regimen（Peg IFN-α-2b+TDF+GM-CSF+HBV vaccine）can improve the rate of HBsAg loss in HBeAg positive na（?）ve CHB patients.And the levels ofΔpgRNA at 24th week in the antiviral course can predict the treatment outcome.For the first time,we use scRNA-seq to systematically describe the changes of immune cells in CHB patients before and after treatment,which provides a new insight for the treatment of CHB.