Occult hepatitis B virus infection in chronic liver disease: full-length genome and analysis of mutant surface promoter

Background and Aims: Genome sequence of hepatitis B virus (HBV) from occult chronic infection is scarce. Fifty-six (9.4%) of 591 patients seronegative for hepatitis B surface antigen (HBsAg) with chronic liver disease were positive for HBV DNA. The complete HBV genome from 9 of these patients (S1-S9) and 5 controls positive for HBsAg (SWT.1-SWT.5) were analyzed. Methods: Overlapping genome fragment amplification, cloning, and sequencing was performed on these cases. Functional analysis of surface promoter was conducted using fusion construct. Results: All patients with occult infection except one (S8) had a low viral titer. Eight patients had infection with genotype A (S1-S5, SWT.1-2, SWT.5) and 6 had infection with genotype D (S6-S9, SWT.3-4). S4 and S5.1 of genotype A had the characteristic nucleotide deletions in core and pre-S1 region seen in genotype D. The major observations in patients with occult HBV infection were as follows: frequent quasispecies variation, deletions in pre-S2/S region affecting the surface promoters (nt 3025-54) and pre-S protein (S3, S5, S6, S8), truncated precore (S6, S8, S7.1) and core (S9) owing to stop signal, alternate start codon for the Polymerase gene (S3, S9), and YMDD mutation (S1, S4, S9) in patients not on antiviral therapy. HBsAg and core proteins could be shown immunohistochemically in 3 of 5 liver biopsy specimens available. The mutant surface promoters (pre-S2 and S) on functional analysis showed alterations in HBsAg expression. Conclusions: These changes in the regulatory region with possible alterations in the ratio of large and small surface proteins along with other mutations in the genome may decrease the circulating HBsAg level synergistically, making the immunodetection in serum negative

Development of highly sensitive bicistronic vector based non-radioactive antigen-specific cytotoxicity assay

In the absence of a better alternate, 51Cr release assay, with its several disadvantages is still the most common method for detection of MHC class I restricted T-cell mediated cytotoxicity. We describe a system in which the T-cell mediated cytotoxicity can be assessed using host-derived cells transfected with a bicistronic vector expressing the specific antigen and a quantifiable reporter as target cells. This overcomes the problems associated with use of radioactivity, pre-loading of target cells with reporter/antigen and the MHC restriction. We used HBV core antigen to prove the concept, as it is an established CTL target. Bicistronic vectors containing HBV core and reporter (EGFP/Fluc) gene were generated and further checked for antigen/reporter expression in human HepG2, mouse fibroblast BALB/c.3T3 and mouse lymphoma A20 cell lines. The effector cells to study the cytolytic activity were generated in vivo using BALB/c mice immunized with antigen expressing DNA clone or protein. The target cells (BALB/c.3T3 and A20) transiently transfected with bicistronic constructs were incubated with effector cells (splenocytes) from immunized mice at a different effector to target ratio. Following incubation the CTL activity was calculated by measuring the reporter luciferase in the remaining viable target cells that inversely correlates with the cytolysis of susceptible cells. The percent specific lysis measured in our assay was compared with conventional 51Cr release assay to validate this approach. This novel bicistronic vector based cytotoxicity assay demonstrated an easy to perform, antigen-specific and non-radioactive method of determining T-cell mediated cytotoxicity