Abstract

Background: Although nucleotide analogs (NA) prevent viral replication in chronic hepatitis B (CHB) patients, it rarely achieve HBsAg seroclearance. While the reduction of HBsAg levels is associated with HBsAg seroclearance, the factors regulating HBsAg levels are not well understood. To aim for “Functional Cure”, we explored the host immunodynamics involved in the reduction of HBsAg levels. Methods: Among CHB patients (HBsAg >1000 IU/mL before the NA treatment) treated with more than five years of NA treatment, we selected four patients whose HBsAg levels decreased below 100 IU/mL (declining group) and four patients whose HBsAg levels maintained above 1000 IU/mL (prolonged group). The peripheral blood mononuclear cells (PBMCs) from these patients, as well as three healthy controls, were sent for single-cell RNA sequencing (scRNAseq) targeting the 400 immune-related genes. PBMCs from additional 32 CHB patients (declining group: n=24, prolonged group: n=8) were used for the validation study. In vitro analysis was performed with HBV-expressing cell line HepG2/2.2.15. Results: The average duration of NA treatment for the eight CHB patients was 9.5 years, and there were no differences in clinical backgrounds including age, gender, type of NA treatment, or duration of treatment between the declining and prolonged groups. A total of 82,000 PBMCs were evaluated through scRNAseq and categorized into 10 immune cell clusters including CD8⁺T cells, CD4⁺T cells, NK cells, B cells, monocytes, and dendritic cells. Although the frequency of each cell population was not different between the declining and prolonged groups, analysis of the differentially expressed gene of each cluster revealed that the IL-1β expression levels of the CD14⁺CD16^- classical monocyte cluster were significantly higher in the declining group than those in the prolonged group. In the validation cohort, the IL-1β expression levels of the isolated circulating CD14⁺ classical monocytes of the declining group were also significantly higher than those of the prolonged group. In vitro analysis, recombinant IL-1β treatment significantly decreased the HBsAg levels in the supernatant of HepG2/2.2.15 in a dose-dependent manner. Conclusion: In CHB patients, the differences in IL-1β production capacity in peripheral blood classical monocytes might be involved in the reduction of serum HBsAg levels during NA treatment.