Introduction: Hepatitis B is the most common blood-borne virus infection and is incurable, requiring long-term suppressive antiviral therapy. Upon treatment cessation, 50-70% will relapse with risk of disease progression, ~30% maintain virological control, and 0-20% clear the infection (loss of HBsAg). Viral persistence is driven by intrahepatic covalently closed circular DNA (cccDNA) and integrated DNA (iDNA) which are challenging to quantify. We aimed to develop novel biomarkers for cccDNA and iDNA to predict relapse or cure (loss of HBsAg). We hypothesise that lower levels of cccDNA and iDNA predict viral clearance.
Methods: We will recruit 130 patients who have been on long-term antiviral treatment with HBV DNA < 20 IU/ml for ≥ 3 years, HBeAg negative, HBsAg < 1000 IU/ml and are older than 18. Before stopping treatment, we will collect liver fine-needle aspirates (FNA) and blood to measure intrahepatic cccDNA, iDNA and peripheral biomarkers (HBV DNA, HBV RNA, HBsAg, ALT and peripheral blood mononuclear cells).
Results: We have obtained baseline FNAs from 15 patients and the procedure was well tolerated, with mild (45%) or moderate (55%) pain reported. Average recovery time was ~48 hours with minimal post-operative care. By staining cells with fluorescently-labelled myrcludex B (a hepatocyte-specific lipopeptide), we found an average of 3.3 x 105 hepatocytes per FNA. We have established highly sensitive PCR-based assays that can measure cccDNA and iDNA levels (requiring ~50 cells as minimum input). Patients have remained well past 6 months post treatment cessation with minimal flares and changes in HBV DNA levels.
Discussion: We will determine how well intrahepatic cccDNA and/or iDNA predict clinical outcomes when stopping antiviral therapy, alongside assessing novel blood biomarkers as surrogate markers for HBV activity and host antiviral immune response. Our results will inform personalised treatment by identifying those who can safely stop treatment and achieve cure.