LAUSR

Background: Occult hepatitis B infection (OBI) is a frequent finding in human immunodeficiency virus (HIV)‐infected patients. While several related mutations in the hepatitis B virus (HBV) genome have been reported, their distinct impact on HBsAg synthesis is largely obscure. Methods: Thirty‐one (18%) out of 172 HIV‐infected patients, who were selected from HBsAg‐negative patients, were positive for HBV‐DNA assigned as being OBI‐positive. We generated a series of expression constructs of variant HBsAg with “a” determinant amino acid substitutions including P127L, P127T, S136Y, and P127T + S136Y using site‐directed mutagenesis. The expression of variant HBsAg was examined by transient transfection in hepatoma cells, followed by HBsAg immunoassay and immunofluorescence stained with specific anti‐HBs antibodies. The potential impact of amino acid substitutions at different positions for conformational changes in the HBsAg was investigated using bioinformatics. Results: All variants comprising either single or combined mutations resulted in significantly reduced HBsAg detection in supernatants and in cell lysates of hepatoma cells transfected with the constructs. Moreover, intracellular immunofluorescence staining of cytoblocks showed perinuclear and cytoplasmic fluorescence of HBsAg constructs with significantly diminished fluorescent intensity in comparison to the wild type. Altered protein conformations by predictive models, indicating an impaired detection by the host's immune response as well as by commercial antibody‐based test assays. Conclusion: Mutations in the “a” determinant region of HBV as often found in OBI remarkably impair the detection of HBsAg from serum and infected cells, emphasizing the relevance of alternative methods such as HBV‐DNA quantification for high‐risk groups like HIV‐infected individuals. J. Med. Virol. 89:246–256, 2017. © 2016 Wiley Periodicals, Inc.