Characterization of Hepatitis B Precore/Core-Related Antigens

Abstract

Current therapies rarely cure chronic hepatitis B virus (HBV) infection due to the persistence of the viral episome, the covalently closed circular DNA (cccDNA), in hepatocytes. The hepatitis B core-related antigen (HBcrAg), a mixture of the viral precore/core gene products, has emerged as one potential marker to monitor the levels and activities of intrahepatic cccDNA. Here, a comprehensive characterization of precore/core gene products revealed that HBcrAg components included the classical hepatitis B core antigen (HBc) and e antigen (HBeAg), and additionally, the precore-related antigen, PreC, retaining the N-terminal signal peptide. Both HBeAg and PreC antigens displayed heterogeneous proteolytic processing at their C-terminus resulting in multiple species, which varied with viral genotypes. HBeAg was the predominant form of HBcrAg in HBeAg-positive patients. Positive correlations were found between HBcrAg and PreC, between HBcrAg and HBeAg, and between PreC and HBeAg, but not between HBcrAg and HBc. Serum HBeAg and PreC shared similar buoyant density and size distributions, and both displayed density and size heterogeneity. HBc, but not HBeAg or PreC antigens, was found as the main component of capsids in DNA-containing or empty virions. Neither HBeAg nor PreC proteins were able to form capsids in cells or in vitro under physiological conditions. In conclusion, our study provides important new quantitative information on levels of each component of precore/core gene products as well as their biochemical and biophysical characteristics, implying that each component may have distinct functions and applications in reflecting intrahepatic viral activities. IMPORTANCE Chronic hepatitis B virus (HBV) infection afflicts approximately 257 million people, who are at high risk of progressing to chronic liver diseases, including cirrhosis, fibrosis, and hepatocellular carcinoma. Current therapies rarely achieve cure of HBV due to the persistence of HBV episome, covalently closed circular DNA (cccDNA), in the nucleus of infected hepatocytes. Peripheral markers of cccDNA levels and transcriptional activities are urgently required to guide antiviral therapy and drug development. Serum hepatitis B core-related antigen (HBcrAg) is one such emerging peripheral marker. We have herein characterized the components of HBcrAg in HBV infected patients as well as in cell cultures. Our results provide important new quantitative information on levels of each HBcrAg component, as well as their biochemical and biophysical characteristics. Our findings further suggest that each HBcrAg component may have distinct functions and applications in reflecting intrahepatic viral activities.

Early Steps of Hepatitis B Life Cycle: From Capsid Nuclear Import to cccDNA Formation

“…Of note, besides the secretion of viral particles that consist of rcDNA-containing virions, different types of incomplete particles are released, particularly empty capsids consisting of genome-free enveloped capsids that are found in infected humans in 100-fold or more excess [ 14 ]. These empty viral particles are currently the subject of research by different groups, as they represent a potential biomarker to monitor intrahepatic HBV activities and the response to antiviral therapies [ 15 , 16 , 17 ]. HBV RNA-containing capsids are also secreted as both non-enveloped and enveloped particles [ 18 , 19 ].…”

Novel Biomarkers of Hepatitis B Virus and Their Use in Chronic Hepatitis B Patient Management

“…Hepatitis B core-related antigen (HBcrAg) is represented by three separate proteins, HBeAg, HBcAg and p22cr ( Figure 1 ), that all share a common 149 amino acid sequence, with the latter being a post-translational product similar to HBeAg but retaining the N-terminal domain [ 39 , 54 ]. HBcrAg levels in serum are measured using a chemiluminescent enzyme immunoassay (CLEIA) processed using the Lumipulse automated analyzer system (Fujirebio Inc.) involving a series of monoclonal antibodies specific to the different protein targets [ 55 ].…”

“…HBeAg is the majority component of HBcrAg (72%), with HBcAg and p22cr both present at approximately 10–15% of total HBcrAg composition in the serum of HBeAg-positive patients [ 58 ]. As mutations in the basal core promotor influence the expression of HBeAg and the presence of anti-HBe antibodies in the serum can also affect HBeAg levels [ 54 ], faulty interpretation of HBcrAg results may occur. In such a scenario, cccDNA levels are unaffected, yet the interpreted relationship between HBcrAg levels and HCC may be distorted [ 54 , 59 ].…”

“…As mutations in the basal core promotor influence the expression of HBeAg and the presence of anti-HBe antibodies in the serum can also affect HBeAg levels [ 54 ], faulty interpretation of HBcrAg results may occur. In such a scenario, cccDNA levels are unaffected, yet the interpreted relationship between HBcrAg levels and HCC may be distorted [ 54 , 59 ]. Furthermore, it has been shown that HBcrAg does not correlate with intrahepatic cccDNA levels in human and chimpanzee serum [ 58 , 60 ], but rather the serum HBcAg component of HBcrAg, primarily comprising empty virions, more closely correlated with intrahepatic cccDNA [ 58 ].…”

Application of the woodchuck animal model for the treatment of hepatitis B virus-induced liver cancer

“…, 10 10 -10 11 particles/mL), while subviral particles containing WHsAg are produced in large excess. Like in human HBV infection[ 69 ], a WHV core-related antigen (WHcrAg), including the classical WHcAg and WHeAg, and additionally, the WHV precore-related antigen (WPreC), is produced during infection in woodchucks, with elevated levels present in chronic WHV carriers[ 57 ]. The high loads of circulating WHeAg and WHsAg produced during chronic WHV infection in woodchucks are thought to be responsible for the immunological tolerance to the virus at the T- and B-cell level[ 30 , 34 , 39 , 40 ], and are further associated with the liver disease progression to chronic hepatitis B and liver cancer[ 31 , 70 , 71 ].…”