The information regarding the effect of hepatitis B virus (HBV) infection on gut microbiota and the relationship between gut microbiota dysbiosis and hepatitis B virus-induced chronic liver disease (HBVCLD) is limited. In this study, we aimed at characterizing the gut microbiota composition in the three different stages of hepatitis B virus-induced chronic liver disease patients and healthy individuals. Faecal samples and clinical data were collected from HBVCLD patients and healthy individuals.The 16S rDNA gene amplification products were sequenced. Bioinformatic analysis including alpha diversity and PICRUSt was performed. A total of 19 phyla, 43 classes, 72 orders, 126 families and 225 genera were detected. The beta-diversity showed a separate clustering of healthy controls and HBVCLD patients covering chronic hepatitis (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC); and gut microbiota of healthy controls was more consistent, whereas those of CHB, LC and HCC varied substantially. The abundance of Firmicutes was lower, and Bacteroidetes was higher in patients with CHB, LC and HCC than in healthy controls. Predicted metagenomics of microbial communities showed an increase in glycan biosynthesis and metabolism-related genes and lipid metabolism-related genes in HBVCLD than in healthy individuals. Our study suggested that HBVCLD is associated with gut dysbiosis, with characteristics including, a gain in potential bacteria and a loss in potential beneficial bacteria or genes. Further study of CHB, LC and HCC based on microbiota may provide a novel insight into the pathogenesis of HBVCLD as well as a novel treatment strategy. K E Y W O R D S16S rDNA, dysbiosis, Gut Microbiota, hepatitis B virus, progression

Pegylated interferon-alpha (PegIFNα) therapy has limited effectiveness in hepatitis B e-antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. However, the mechanism underlying this failure is poorly understood. We aimed to investigate the influence of bile acids (BAs), especially taurocholic acid (TCA), on the response to PegIFNα therapy in CHB patients. Here, we used mass spectrometry to determine serum BA profiles in 110 patients with chronic HBV infection and 20 healthy controls (HCs). We found that serum BAs, especially TCA, were significantly elevated in HBeAg-positive CHB patients compared with those in HCs and patients in other phases of chronic HBV infection. Moreover, serum BAs, particularly TCA, inhibited the response to PegIFNα therapy in HBeAg-positive CHB patients. Mechanistically, the expression levels of IFN-γ, TNF-α, granzyme B, and perforin were measured using flow cytometry to assess the effector functions of immune cells in patients with low or high BA levels. We found that BAs reduced the number and proportion and impaired the effector functions of CD3+CD8+ T cells and natural killer (NK) cells in HBeAg-positive CHB patients. TCA in particular reduced the frequency and impaired the effector functions of CD3+CD8+ T and NK cells in vitro and in vivo and inhibited the immunoregulatory activity of IFN-α in vitro. Thus, our results show that BAs, especially TCA, inhibit the response to PegIFNα therapy by impairing the effector functions of CD3+CD8+ T and NK cells in HBeAg-positive CHB patients. Our findings suggest that targeting TCA could be a promising approach for restoring IFN-α responsiveness during CHB treatment.

The mechanism for the co-existence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in chronic HBV infected patients remains controversial. This study aimed to explore the role of HBV S gene mutation and anti-HBs subtype-nonspecificity in patients with simultaneous HBsAg/anti-HBs positivity. Chronic HBV infections with (n = 145, group I) and without (n = 141, group II) anti-HBs were included. The S gene was amplified and sequenced. The neutralization experiment was used in group I patients' sera to determine the specificity of anti-HBs. Additionally, the HBV vaccinated persons' sera were used to estimate the neutralize capacity of anti-HBs against HBsAg in group I patients. Results showed that 2.63% (145/5513) chronic HBV infected patients had positive results for anti-HBs. HBsAg amino acid (aa) substitution rate in 35 patients of group I was significantly higher than that in 58 patients of group II (1.89% vs 0.95%, P < 0.05), especially within "a" determinant (4.05% vs 1.22%, P < 0.05). In group I patients, anti-HBs in (74.29%, 26/35) patients was not directed to the subtypes of the co-existing HBsAg. Besides, some HBsAg variations in group I patients, sG145R mutation, inserted mutations, and continuous aa mutations within the major hydrophilic region (MHR), decreased the neutralized capacity of anti-HBs from HBV vaccinated persons. In conclusion, both of HBsAg mutation and anti-HBs subtype-nonspecificity contributed to the co-existence of HBsAg and anti-HBs in chronic HBV infection. HBV vaccine recipients may still have a risk of HBV infection when exposure to patients with simultaneous HBsAg/anti-HBs positivity.

NTCP gene polymorphisms may be associated with the natural course of HBV infection in a Chinese Han population. The S267F variant may be a protective factor to resist chronic hepatitis B progression which showed a higher bile acid level in Chinese Han chronic HBV infection patients. The rs4646285 variants could influence the expression of NTCP at the level of transcription, and ultimately may be associated with HBV infection immune recovery.