Molecular characterization of hepatitis B virus surface antigen mutants in Singapore patients with hepatocellular carcinoma and hepatitis B virus carriers negative for HBsAg but positive for anti-HBs and anti-HBc

Background and Aims: Mutations on the a-determinant of hepatitis B virus surface antigen (HBsAg), capable of escaping detection and vaccination, are identified in HBsAg-positive/anti-HBs-positive vaccinated infants. We studied the prevalence of these mutants in HBsAg-negative/anti-HBc-positive chronic HBV carriers and patients with hepatocellular carcinoma (HCC). Methods: DNA sequence coding for the antigenic a-determinant of HBsAg was amplified from either HCC genomic DNA or serum samples of the selected patients and sequenced. The replicative mutant genomes were reconstituted in vitro and their reactivity to commercial kits measured. Results: Mutations within and/or outside the a-determinant were identified in patients seronegative for HBsAg. They were then reconstituted in vitro and transiently transfected into HepG2 cells. Culture medium containing secreted HBV viral particles was collected and assayed for their binding to commercial kits. Drastic decrease of reactivity to these kits was seen with most of the identified mutations, including those located outside the a-determinant. Conclusion: The existence of a more complex antigenic structure of HBsAg is indicated by the decreased reactivity to detection of mutations, some of which are outside the a-determinant, escape vaccination and may persist in seronegative patients. The high proportion of HBsAg mutants that are integrated in HCC genomes suggests a role of these mutants in hepatocarcinogenesis, possibly leading to mutant HBV-related HCC.