. Chronic hepatitis B virus (HBV) infection has long remained a critical global health issue. Covalently closed circular DNA (cccDNA) is a persistent form of the HBV genome that maintains HBV chronicity. Decades of extensive research resulted in the two therapeutic options currently available: nucleot(s)ide analogs and interferon (IFN) therapy. A plethora of reliable markers to monitor HBV patients has been established, including the recently discovered encapsidated pregenomic RNA in serum, which can be used to determine treatment end-points and to predict the susceptibility of patients to IFN. Additionally, HBV RNA splice variants and cccDNA and its epigenetic modifications are associated with the clinical course and risks of hepatocellular carcinoma (HCC) and liver fibrosis. However, new antivirals, including CRISPR/Cas9, APOBEC-mediated degradation of cccDNA, and T-cell therapies aim at completely eliminating HBV, and it is clear that the diagnostic arsenal for defining the long-awaited sterilizing cure is missing. In this review, we discuss the currently available tools for detecting and measuring HBV RNAs and cccDNA, as well as the state-of-the-art in clinical implications of these markers, and debate needs and goals within the context of the sterilizing cure that is soon to come.

Hepatitis B virus (HBV) infection can be managed clinically with nucleos(t)ide therapy, which suppresses viral replication; however, these drugs must often be used long term, as they are unable to fully eliminate the virus. For many patients, discontinuation of treatment results in viral resurgence and hepatic flare, and there is not a reliable way to identify those individuals that can be successfully taken off nucleos(t)ide therapy. In this issue of the JCI, Rivino and colleagues report on their use of a multipronged approach to investigate potential biomarkers indicative of HBV-infected patients who can safely stop nucleos(t)ide therapy. The authors identified a population of HBV-specific, PD1-positive T cells that was present in HBV-infected patients who successfully discontinued treatment without hepatic flare, but not in those that developed flare upon treatment cessation. Together, these results support the concept that PD1+ cells may play an important role in viral control, the further evaluation of this T cell subset in preventing hepatic flare, and the development of assays to better detect this PD1+ T cell population in HBV-infected patients on nucleos(t)ide therapy.

Although the efficacy of nucleos(t)ide analogue (NA) has been confirmed for treatment of chronic hepatitis B, long-term therapy has been recommended due to the high frequency of off-therapy viral DNA rebound and disease relapse. In this review, the RNA virion-like particles of hepatitis B virus (HBV) are integrated into the life cycle of HBV replication, and the potential significance of serum HBV RNA is systematically described. The production of HBV RNA virion-like particles should not be blocked by NA; in this regard, serum HBV RNA is found to be a suitable surrogate marker for the activity of intrahepatic covalently closed circular DNA (cccDNA), particularly among patients receiving NA therapy. Therefore, the concept of virological response is redefined as persistent loss of serum HBV DNA and HBV RNA. In contrast to hepatitis B surface antigen (HBsAg) that can originate from either the cccDNA or the integrated HBV DNA fragment, serum HBV RNA, with pregenomic RNA origination, can only be transcribed from cccDNA. Therefore, the loss of serum HBV RNA would likely be a promising predicator for safe drug discontinuation. The clinical status of consistent loss of serum HBV RNA accompanied with low serum HBsAg levels might be implicated as a "para-functional cure," a status nearly close to the functional cure of chronic hepatitis B, to distinguish the "functional cure" characterized as serum HBsAg loss with or without anti-HBs seroconversion.