Rapid whole-blood immune profiling reveals heterogeneous HBV-specific T cell response patterns independent of clinical disease phases

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.