BACKGROUND:Finite therapy resulting in sustained hepatitis B surface antigen (HBsAg) loss for patients with chronic HBV infection (CHB) is an important therapeutic goal. In patients with HBeAg-negative infection, nucleos(t)ide analog (NA) withdrawal may achieve HBsAg loss in 5%-20% of patients after 3 years. Pegylated interferon (PEG-IFNα) is a recognized treatment for CHB. METHODS:NUC-B was a randomized, multicenter trial in NA-treated non-cirrhotic HBeAg-negative patients with CHB. Patients were allocated to either NA withdrawal alone (control) or NA withdrawal followed by a 16-week course of PEG-IFNα 180 μg weekly commencing 4 weeks after NA cessation (PEG-IFNα). The primary endpoint was HBsAg loss at 3 years. RESULTS:The target recruitment of 240 patients was not achieved. In all, 156 patients, 82 to the control arm, 74 to the PEG-IFNα arm, were recruited between 2017 and 2021; median age 45 years, 24% female, HBV genotypes-A 16%, B 6%, C 4%, D 24%, E 22%, other 1%, ānd unknown 26%. At 3 years, 3% of patients in the control arm and 14% of patients in the PEG-IFNα arm lost HBsAg (OR 5.39; 95% CI (1.11, 26.19); p =0.037). In the control arm, 34.9% of patients returned to NA therapy compared with 28.4% in the PEG-IFNα arm. Exaggerated flares occurred in 27.9% of patients in the control arm compared with 13.4% in the PEG-IFNα arm. CONCLUSIONS:The use of adjuvant PEG-IFNα therapy after withdrawal of NA therapy increases the rate of HBsAg loss while simultaneously reducing the number of exaggerated flares.

Background & Aims: Virus-specific T cells are crucial for controlling viral replication and disease progression in chronic hepatitis B (CHB). Nevertheless, due to the complexity of their analysis, CHB management relies exclusively on virological and biochemical markers. We aimed to develop a point-of-care immunological test, the HBV-cytokine release assay (HBV-CRA), designed to measure multiple cytokines in whole blood after HBV-peptide pool stimulation. Methods: We first assessed the assay′s sensitivity by spiking whole blood with engineered HBV-specific T cells. Next, the performance of HBV-CRA was compared to ex vivo IFN-γ ELISpot assays in terms of sensitivity and consistency. We then applied the assay in a cross-sectional study of 235 CHB patients and longitudinally in acute HBV patients during HBsAg sero-clearance. Results: The HBV-CRA detected cytokine secretion in response to peptide pools in approximately 80% of CHB patients. A profile of high IL-2 and IFN-γ after Core peptide stimulation was significantly associated with HBsAg clearance in acute HBV. The assay also revealed variability in cytokine levels and responses to different HBV proteins, even within the same clinical phase of CHB. Unsupervised clustering identified distinct HBV-specific immune patterns that did not segregate by clinical and virological categories. Notably, the assay revealed that NUC treatment impacted the functional profile of HBV-specific T cell responses. Conclusions: The HBV-CRA is a rapid, easy-to-use assay that detects immune profiles linked to HBsAg clearance and distinguishes CHB patients based on antiviral T cell function. This capability to stratify patients within similar clinical phases offers a novel biomarker for host-viral interactions and could guide the selection of novel immunotherapies. ### Competing Interest Statement NLB, ATT, and AB are the founders and hold stocks of T Cell Diagnostics (TCD), a biotech company that is developing T cell tests. SKH is an employee of TCD. None of the other authors have any relevant conflict of interest.

BACKGROUND & AIMS:There is an unmet need for immunological biomarkers in chronic HBV infection (CHB), where patient management relies on virological and biochemical markers despite the crucial role of virus-specific T cells in controlling viral replication and disease progression. Here, we developed the HBV-cytokine release assay (HBV-CRA), a rapid, point-of-care test, to define whether HBV-specific T cell functional patterns are linked with conventional disease phase classifications. METHODS:Peptides covering pan-genotype HBV proteomes were utilized to trigger cytokine release by HBV-specific T cells in whole blood. We first assessed the assay's sensitivity by spiking whole blood with engineered HBV-specific T cells. Next, we compared the sensitivity and reproducibility of the HBV-CRA to ex vivo IFN-γ ELISpot assays. We then applied the assay in a cross-sectional study of 235 patients with CHB and longitudinally in patients with acute HBV infection (AHB) during HBsAg seroclearance. RESULTS:The HBV-CRA detected T cell function in 80% of CHB cases and showed that elevated IL-2 and IFN-γ levels after Core peptide stimulation were associated with HBsAg clearance in AHB. Unsupervised clustering identified distinct immune response patterns independent of established clinical and virological classifications and detected a functional impact of NUC treatment on HBV-specific T cell responses. CONCLUSIONS:The HBV-CRA is an easy-to-use assay that identifies immune profiles associated with HBsAg clearance in AHB and differentiates patients with CHB based on antiviral T cell function. Importantly, distinct HBV-specific T cell cytokine patterns were detected independently of conventional clinical disease phases, suggesting that stratification of patients with CHB for immunotherapeutic interventions should be guided by the HBV-CRA. IMPACT AND IMPLICATIONS:Challenges in collecting anti-HBV immune biomarkers from patients with chronic HBV infection (CHB) have led us to develop an easy-to-use assay (HBV-CRA) designed to quantify virus-specific T cell function in patient whole blood. We show that the HBV-CRA can identify immune profiles associated with HBsAg clearance in AHB and reveal functional T cell heterogeneity among patients with CHB that is not captured by standard clinical classifications. By offering a scalable, point-of-care immune monitoring tool, the HBV-CRA could support the development and implementation of personalized immunotherapeutic strategies in CHB management.

As new evidence emerges, treatment strategies toward the functional cure of chronic hepatitis B are evolving. In 2019, a panel of national hepatologists published a Consensus Statement on the functional cure of chronic hepatitis B. Currently, an international group of hepatologists has been assembled to evaluate research since the publication of the original consensus, and to collaboratively develop the updated statements. The 2.0 Consensus was aimed to update the original consensus with the latest available studies, and provide a comprehensive overview of the current relevant scientific literatures regarding functional cure of hepatitis B, with a particular focus on issues that are not yet fully clarified. These cover the definition of functional cure of hepatitis B, its mechanisms and barriers, the effective strategies and treatment roadmap to achieve this endpoint, in particular new surrogate biomarkers used to measure efficacy or to predict response, and the appropriate approach to pursuing a functional cure in special populations, the development of emerging antivirals and immunomodulators with potential for curing hepatitis B. The statements are primarily intended to offer international guidance for clinicians in their practice to enhance the functional cure rate of chronic hepatitis B.