Dynamics of Serum Hepatitis B Virus Nucleic Acids in Patients With Chronic Hepatitis B After Stopping Nucleos(t)ide Analogs Reveal Delayed Rebound of Hepatitis B Virus RNA

Serum HBV RNA, an emerging biomarker, was reported to require a longer duration than HBV DNA to achieve undetectable levels during nucleos(t)ide analogs (NAs) therapy according to the analysis of viral nucleic acid kinetics. However, detailed information about the viral dynamics of off-treatment remains unclear. A total of 32 hepatitis B e antigen (HBeAg)-positive patients who stopped telbivudine-based therapy after meeting stopping criteria were included. Laboratory assays were tested every 12 or 16 weeks. The extracellular and intracellular viral dynamics in HepAD38 cells were monitored during and after NA exposure. NA therapy induced a significantly faster decline of serum HBV DNA than serum HBV RNA (slope, -0.1103 vs. -0.0459, p < 0.0001). After treatment cessation, serum HBV DNA rebounded more rapidly than serum HBV RNA during virological relapse (slope, 0.1752 vs. 0.1142, p = 0.0184). The peak rebound amplitude of serum HBV DNA occurred within the first 12 weeks off-treatment, significantly surpassing that of serum HBV RNA [0.24 (0.12, 0.46) vs. 0 (0, 0.10), p < 0.0001]. During the off-treatment phase in HepAD38 cells, different from the strong rebound of extracellular HBV DNA (56.40-fold, p < 0.0001) with faster rebound kinetics (slope, 0.1479 vs. 0.0249, p < 0.0001), extracellular HBV RNA increased only 2.65-fold (p = 0.0452). This occurred despite a significantly faster rebound of intracellular HBV RNA compared to HBV DNA (0.0106 vs. 0.2508, p < 0.0001). In conclusion, serum HBV RNA rebounded later and less prominently than serum HBV DNA after NAs cessation. The HepAD38 model indicated preferential intracellular HBV RNA utilization for HBV DNA synthesis and secretion upon reverse transcription reactivation.