Virologic And Immunological Factors Associated With Treatment Efficacy Of Switching From Nucleos(t)ide Analogue To Interferon In Virally-suppressed Chronic Hepatitis B Patients

Background&Aims:There remains 240 million people with chronic hepatitis B virus（HBV）infection worldwide,which causes 887 000 mortality per year.Hepatitis B surface antigen（HBsAg）loss is considered a functional cure and the ideal endpoint of antiviral therapy for chronic hepatitis B（CHB）.However,it is estimated that 52.2 years would be needed to clear HBsAg by nucleos（t）ide analogue（NA）treatment.Sequential pegylated interferon alpha（Peg-IFN-α）treatment in virally suppressed CHB patients undergoing long-term NA therapy significantly increases the rate of HBsAg loss,and functional cure can be sustained in a proportion of patients who lose HBsAg after Peg-IFN-αdiscontinuation.To date,the virologic and immunological kinetics associated with Peg-IFN-αinduced HBsAg loss,in particular durable functional cure post therapy,are still unknown.In addition,virologic breakthrough（VBT）may occur after switching from NA to Peg-IFN-αmonotherapy and result in treatment failure.This study evaluated the longitudinal changes in virologic and immunological biomarkers during and post sequential Peg-IFN-αtherapy in NA-suppressed CHB patients,aiming to identify biomarkers associated with treatment response,explore the predictive model of durable functional cure after Peg-IFN-αbased therapy,and dissect the potential mechanisms of VBT.Methods:Of 257 NA-suppressed CHB patients in the ANCHOR study（NCT02327416）,80patients randomly assigned to 96-week Peg-IFN-αbased therapy with 24-week off-treatment follow-up from March 2014 to June 2019 were included in Study 1 and Study 2.VBT was defined as HBV DNA level increased by more than 1 log₁₀ IU/m L from the nadir during treatment.A response was defined as HBsAg loss and/or hepatitis B surface antibody（HBsAb）appearance at the end of treatment（EOT）.Sustained response（SR）or durable functional cure was defined as sustained HBsAg loss at the end of follow-up（EOF）.Blood samples were drawn at baseline and every 24 weeks until EOF to monitor the dynamic changes in virologic parameters.Paired liver biopsies were performed at baseline and week48 to determin intrahepatic HBV covalently closed circular DNA（cccDNA）.Immune responses including HBV specific CD8⁺T cells,follicular helper T（Tfh）cells and B lymphocytes proportions were examined at EOT and EOF.A total of 166 NA-suppressed CHB patients receiving sequential Peg-IFN-αtherapy from Tongji Hospital were included in Study 3.Thity-six patients received Peg-IFN-α（180μg/week）in combination with 0.5 mg ETV daily for 8 weeks followed by Peg-IFN-αmonotherapy for 40 weeks between April 2009 and December 2011（Cohort 1）,130 patients received Peg-IFN-αin combination with ETV for 48 weeks followed by Peg-IFN-αmonotherapy for 48 weeks between March 2014 and December 2019（Cohort 2）.Innate and adaptive immune cell proportions were examined dynamically in peripheral blood every 12or 24 weeks.Liver biopsy specimens were collected at baseline and week 48.Ex vivo effect of IFN-αon the expressions of Toll-like receptor 2（TLR2）and programmed cell death ligand 1（PDL1）on monocytes,programmed cell death receptor 1（PD1）on CD8⁺T cells was examined.Peripheral blood mononuclear cells（PBMCs）were treated with TLR2agonist and/or PDL1 blockade to evaluate the production of cytokines,function of CD8⁺T cells,and their effect on HBV DNA replication.Results:Of the 80 patients enrolled in Study 1 and Study 2,36 achieved a response at EOT.Responders were profoundly younger and had lower baseline HBsAg level than nonresponders(2.652±0.072 vs 2.915±0.058 log₁₀ IU/m L,P=0.005).Compared with nonresponders,responders had greater reductions in HBsAg and hepatitis B core-related antigen（HBcrAg）levels at all time points during therapy.From baseline to week 48,a significant decrease in cccDNA level was observed only in responders（0.279±0.072 vs0.131±0.042 copies/cell,P=0.003）.A model combining age,baseline HBsAg level and HBsAg decline from baseline to week 24 can effectively predict treatment response with an AUROC of 0.924（0.864-0.984,P<0.001）,and the AUROC can be improved to 0.938（0.879-0.997,P<0.001）when further combined with the HBcrAg level at week 24.Of the 36 responders,21 maintained SR at EOF.From baseline to week 48,only sustained responders exhibited significantly decreased intrahepatic cccDNA level（0.294±0.109 vs 0.088±0.033 copies/cell,P=0.007）.Compared with nonsustained responders,sustained responders had profoundly lower levels of HBsAg and HBcrAg,but significantly higher level of HBsAb at EOT.Quantitative levels of HBcrAg and HBsAb at EOT were independently correlated with SR.The AUROC was 0.697（0.512-0.882,P=0.047）and 0.744（0.573-0.915,P=0.013）,respectively.A combination of HBcrAg<4log₁₀ U/m L and HBsAb>2 log₁₀ IU/L at EOT had a positive predictive value of 100%for SR with an AUROC of 0.822（0.684-0.961,P=0.001）.These patients showed maintained proportions of HBV envelope-specific CD8⁺T cells and B lymphocytes,a markedly increased proportion of Tfh cells after Peg-IFN-αdiscontinuation,and significantly higher proportions of HBV polymerase-specific CD8⁺T cells and CD86⁺CD19⁺B lymphocytes at EOF.Of the 166 patients enrolled in Study 3,all patients achieved viral suppression before NA cessation.Thirty-three patients switching to Peg-IFN-αexperienced VBT after NA cessation,with majority being hepatitis B e antigen（HBe Ag）positive or having higher HBcrAg level at baseline.Patients with VBT exhibited lower proportions of TLR2⁺monocytes and increased PD1⁺HBV-specific CD8⁺T cells during the early phase of Peg-IFN-αtherapy after NA cessation in peripheral blood,as well as fewer TLR2⁺CD68⁺macrophages but more PDL1⁺CD68⁺macrophages and PD1⁺CD8⁺T cells in liver tissues.Upon IFN-αstimulation ex vivo,PDL1⁺monocytes and PD1⁺CD8⁺T cells were upregulated,whereas TLR2⁺monocytes were not increased in HBe Ag-positive patients,or those with high HBcrAg titers(>5 log₁₀ U/m L).TLR2 stimulation ex vivo induced a significant increase in（Interleukin,IL）-1β,IL-2,IL-4,IL-6,IL-8,IL-10,IL-12p70,IL-13,（Tumor necrosis factor,TNF）-αand IFN-γproduction from PBMCs.Particularly,HBe Ag-positive patients exhibited a less profound IL-6 but more potent IL-10 production,when compared with HBe Ag-negative patients.Blockade of PDL1/PD1 pathway pronouncedly enhanced the expression of CD107a,Perforin,Granzyme B and IFN-γof CD8⁺T cells,especially in HBe Ag-negative patients.Concomitant use of TLR2 agonist and PDL1 blockage suppressed HBV DNA replication by promoting cytokines production and CD8⁺T cells cytotoxicity.Conclusions:1.The age,HBsAg and HBcrAg levels at the early stage of therapy can predict HBsAg clearance and/or HBsAb appearance induced by Peg-IFN-αbased treatment.2.The end-of-treatment HBcrAg<4 log₁₀ U/m L and HBsAb>2 log₁₀ IU/L,proposed as HBVCure cr Ab model,reflect lower intrahepatic cccDNA level,more stable HBV-specific cytotoxic T lymphocyte proportions,as well as sustained Tfh and B cell immune responses.This model can be used to identify patients likely to achieve a durable functional cure post Peg-IFN-αbased treatment.3.Lower expression of TLR2 on monocytes and the enhancement of the PDL1/PD1signaling pathway between monocytes and CD8⁺T cells potentially contribute to VBT after switching from NA to Peg-IFN-α.This insufficient anti-HBV immunity may be partially associated with the HBe Ag serostatus and HBcrAg level.