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Little is known for factors associated with virologic breakthrough (VBT) after switching from nucleos(t)ide analogue (NA) to pegylated interferon alpha (Peg-IFN-α) for patients with chronic hepatitis B (CHB). Eighty patients who received 48-week Peg-IFN-ɑ and NA combination therapy followed by Peg-IFN-ɑ monotherapy for additional 48 weeks were included in this study. HBV-related markers including HBV DNA, HBsAg, HBcrAg, HBeAg, cccDNA, and immunological biomarkers were dynamically evaluated. Twelve (15.0%) patients experienced VBT after switching to Peg-IFN-ɑ and exhibited significantly lower rates of HBsAg loss after therapy completion (0% vs. 35.3%, p = 0.014). The patients with HBcrAg≥ 5 log 10 U/mL and HBsAg≥ 100 IU/mL had the highest risk of VBT and failed to achieve subsequent HBsAg clearance. Intrahepatic cccDNA level was significantly higher in patients with HBcrAg≥ 5 log 10 U/mL than those with HBcrAg< 5 log 10 U/mL. Notably, in contrast to patients with HBcrAg< 5 log 10 U/mL or with HBsAg< 100 IU/mL who had obviously restored HBV-specific CD8 + T cell, Tfh or B cell responses before NA cessation, those with HBcrAg≥ 5 log 10 U/mL or with HBsAg≥ 100 IU/mL exhibited lackluster immunities before NA cessation and notable diminished immune responses thereafter. Monitoring HBcrAg and HBsAg levels, which correlated with poor immune responses during sequential Peg-IFN-ɑ strategy, may help to avoid VBT and improve functional cure of CHB., (© 2024 Wiley Periodicals LLC.)

Serum hepatitis B virus (HBV) RNA is a new serological indicator reflecting viral replication with good clinical application prospects. This study aimed to clarify the dynamic changes of serum HBV RNA levels and the quasispecies of HBV RNA virus-like particles in nucleos(t)ide analogues (NAs)-experienced chronic hepatitis B (CHB) patients harboring NAs-resistant mutations and their identifiable effects on NAs resistance. We included CHB patients who were on long-term NAs treatment and with HBV DNA rebound. The longitudinally dynamics of serum HBV RNA levels were quantitatively detected, and the quasispecies differences between serum HBV DNA and serum HBV RNA were compared by high-throughput sequencing. The effect of NAs concentration pressure on altering the resistance mutations quasispecies proportion of HBV DNA and HBV RNA in cell supernatant was analyzed in vitro. A total of 447 serum samples from 36 CHB patients treated with NAs were collected. The median follow-up period was 47 months (about 4 years), and the longest follow-up period was 117 months (about 10 years). Our results showed that HBV RNA could reflect virological breakthrough in 23 (64%, 23/36) patients, and serum HBV RNA rebound earlier than HBV DNA in 12 (52%, 12/23) patients. However, serum HBV RNA remained at a consistently high level and did not fluctuate significantly with the HBV DNA rebound in 6 of 36 patients. In addition, serum HBV RNA was not consistently detectable in 7 of the 36 patients, and their serum HBV RNA was undetectable even after HBV DNA had rebounded. The proportion of drug-resistant mutations in HBV DNA was higher than that of HBV RNA by high-throughput sequencing. The results of in vitro experiments showed that the viral strains with drug-resistant mutation in HBV DNA in cell supernatants gradually become the dominant strains with the increase of NAs concentrations. Serum HBV RNA levels can reflect virological breakthrough in most NAs- treated CHB patients, but there are certain limitations. NAs alter the quasispecies composition of serum HBV DNA and serum HBV RNA, resulting in a higher detection rate of drug-resistant mutations in serum HBV DNA than in serum HBV RNA., (© 2023 Wiley Periodicals LLC.)