Heterogeneous T cell responses across Hepatitis B virus clinical phases revealed by rapid whole-blood HBV T cell analysis

Background & Aims There is an unmet need for immunological biomarkers in chronic hepatitis B (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. To address this, we developed the HBV-cytokine release assay (HBV-CRA), a rapid, point-of-care test that measures multiple cytokines in whole blood after HBV-peptide stimulation.

We first assessed the assay’s sensitivity by spiking whole blood with engineered HBV-specific T cells. Next, we compared sensitivity and consistency of HBV-CRA to ex vivo IFN-γ ELISpot assays. We then applied the assay in a cross-sectional study of 235 CHB patients and longitudinally in acute HBV patients during HBsAg sero-clearance.

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. Importantly, unsupervised clustering identified distinct immune response patterns independent of established clinical and virological classifications. The assay also demonstrated the functional impact of NUC treatment on HBV-specific T cell responses.

The HBV-CRA is a rapid and easy-to-use assay that identifies immune profiles associated with HBsAg clearance and differentiates CHB patients based on antiviral T cell function. Its application in a large CHB cohort revealed that traditional disease phase classifications, based on viral and clinical parameters, cannot predict HBV-specific T cell profiles. The HBV-CRA has the potential to guide patient stratification for immunotherapeutic interventions. 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. Footnotes Conflicts of interest: 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. Funding: This work was supported by the National Medical Research Council (NMRC) Singapore Translational Research Investigator Award (STaR) MOH-001633 (STaR24jan-0004) to AB, an Italian Ministry of Foreign Affairs and International Cooperation (MAECI) and the Agency for Science, Technology and Research (A*STAR) First Executive Programme of Scientific and Technological Cooperation Grant (R22I0IR119) to AB and PL and a Joint Centre for Technology & Development grant (JCTED-ACE-22-01) from Duke-NUS Medical School to NLB and WCC. HBV clincial phases were revised for some CHB patients.