Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA

Kuan-hui Xiang, Eleftherios Michailidis, Hai Ding, Ya-qin Peng, Ming-ze Su, Yao Li, Xue-en Liu, Viet Loan Dao Thi, Xian-fang Wu, William M. Schneider, Charles M. Rice, Hui Zhuang, Tong Li

As important virological markers, serum HBsAg and HBV DNA levels show large fluctuations among chronic hepatitis B (CHB) patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid (AA) substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type, WT) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 AAs, of which 104 (46.02%) had mutations in our study. Some mutations (e.g. sE2G, sL21R, sG24K, sT47A/K, sC69stop (sC69(∗)), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r = 0.61, p < 0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69(∗), and sG145R by co-expression of WT HBsAg. The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements. HBsAg is a major viral protein of hepatitis B virus (HBV) secreted into patient serum and its quantification value serves as an important virological marker for the evaluation of chronic HBV infection and antiviral response. We found a few novel amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These different substitutions might impair virion secretion, change HBsAg antigenicity or impact HBV replication, which all could result in decreased detectable levels of serum HBsAg. The factors affecting circulating HBsAg level and HBsAg detection are varied and caution is needed when interpreting clinical significance of serum HBsAg levels.