Jasper zu Putlitz

Antisense RNA complementary to hepatitis B virus specifically inhibits viral replication

BACKGROUND & AIMS Chronic infection with the hepatitis B virus (HBV) is a major public health problem, and currently available therapies have limited efficacy. Gene therapy strategies for HBV infection are under active investigation. We evaluated the potential of antisense RNA transcribed from antisense genes to interfere with HBV replication. METHODS Subgenomic fragments of the HBV genome were studied with respect to the property of inhibiting HBV replication when intracellularly expressed in the antisense orientation. RESULTS Antisense RNAs derived from the HBV genome specifically inhibited HBV replication and antigen expression in human hepatocellular carcinoma cells by 60%-75%. DNA sequences corresponding to the identified RNAs had no effect on HBV replication, indicating that inhibitory effects are mediated by RNA. Transcripts corresponding to the inhibitory subgenomic fragments were present at high levels. One antisense RNA was found to reduce the amount of pregenomic RNA encapsidated into core particles as a molecular mechanism of antiviral effects. CONCLUSIONS Certain antisense RNA molecules will have substantial antiviral effects against HBV. Antisense RNAs derived from the HBV genome are promising candidates as antiviral agents and may serve as novel tools to identify functionally important regions of HBV transcripts.

Nucleic acid-based antiviral and gene therapy of chronic hepatitis B infection

Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. There is a need to develop new antiviral approaches for the treatment of this disease. We have explored various nucleic acid‐based strategies designed to inhibit HBV replication including: the use of antisense RNA and DNA constructs, DNA‐based immunization techniques to stimulate broad‐based cellular immune responses with particular emphasis on the generation of cytotoxic lymphocyte (CTL) activity to viral structural proteins, hammerhead ribozymes to cleave HBV pregenomic RNA in vitro and dominant negative HBV core mutant proteins as inhibitors of nucleocapsid formation within cells. In order to optimize these antiviral effects, various novel expression vectors have been developed to deliver such DNA constructs to cells. For example, adenoviral vectors carrying genes that encode for dominant negative proteins have been employed to transfect hepatocytes in vitro and in vivo. In addition, plasmid vectors have been produced to promote expression of HBV structural genes following injection into muscle cells as a means to stimulate the hosts cellular and humoral immune response in the context of histocompatibility antigen (HLA) class I and II antigen presentation. These experimental approaches may have important implications for the generation of efficient antiviral effects during chronic HBV infection.

Theoretical and experimental selection parameters for HBV-directed antisense RNA are related to increased RNA-RNA annealing.

Annealing kinetics of antisense species against two different target regions of the hepatitis B virus (HBV) were measured by kinetic in vitro selection. Individual association rates were related to energies calculated for local sequence segments and predicted structures of the complete pregenomic target RNA. A relationship between the presence of external loops and joint sequences with fast pairing was observed whereas internal loops did not favor fast RNA-RNA annealing. The findings were used to predict a fast-annealing HBV-directed antisense oligodeoxyribonucleotide that turned out to pair with its target RNA at an association rate constant of k=9.2 x 10(4) M(-1) s(-1), which is substantially faster than the annealing rates of artificial antisense RNA so far included in in vitro selection assays.

Cytotoxic T cell responses against hepatitis B virus polymerase induced by genetic immunization

BACKGROUND/AIMS Individuals with chronic hepatitis B may benefit from genetic (DNA-based) immunization through induction of viral clearance by enhancement of suboptimal cellular immune responses. While marked cellular immune responses to hepatitis B virus (HBV) nucleocapsid and envelope proteins occur after genetic immunization in mice, it is unknown whether genetic immunization is capable of eliciting such responses to HBV polymerase. We wished to develop assays for the determination of HBV polymerase specific immune responses in mice and investigate whether genetic immunization may elicit humoral and cellular immune responses to HBV polymerase. METHODS BALB/c (H-2d) mice were injected with a DNA expression construct for HBV polymerase. Humoral immune responses to HBV polymerase were analyzed with a newly established ELISA. Cellular immune responses were determined using recombinant vaccinia virus infected target cells expressing HBV polymerase at high levels. RESULTS Assays for the detection of HBV polymerase-specific immune responses were developed. Immunized animals exhibited substantial polymerase-specific cytotoxic T lymphocyte responses. However, no humoral immune responses to HBV polymerase were detectable. CONCLUSIONS Our study demonstrates that DNA-based immunization will generate substantial CTL responses to HBV polymerase and may be an important component of an immunotherapeutic strategy to combat chronic HBV infection.

Cloning, bacterial synthesis, and characterization of immunoglobulin variable regions of a monoclonal antibody specific for the hepatitis B virus X protein.

The nucleotide (nt) sequences encoding the variable regions of the heavy (H) and light (L) chains were determined for a murine monoclonal antibody, 12/231/93, which is specific for a linear epitope located between amino acids 90 and 102 of the hepatitis B virus (HBV) X protein (HBx). The variable (V) regions of the H and L chains were shown to belong to the mouse H chain subgroup II (C) and kappa L chain group III, respectively. The cloned variable region sequences were used for the production of a Fab fragment in Escherichia coli, which had binding activity for membrane immobilized recombinant HBx. These gene sequences may be useful for the study of HBx function in cells that will support HBV replication.

Hepatitis B virus replication and viral antigen synthesis in hepatocyte lines derived from normal human liver

Transient transfection and in vitro infection experiments were performed to characterize replication and antigen synthesis of the hepatitis B virus (HBV) in human hepatocyte lines HH29 and HHY41, derived from normal liver tissue. These liver cell lines are capable of supporting HBV replication and gene expression at levels similar to the human hepatoma cell line HuH-7. Strikingly, a very tight adhesion of HBV to the outer cell membrane of HH29 and HHY41 was observed under conditions that removed HBV to undetectable levels from HuH-7 hepatoma cells. However, no productive HBV infection could be established in these cells as determined by the absence of viral transcripts and de novo antigen synthesis. In conclusion, the human hepatocyte cell lines HH29 and HHY41 may be useful to study important aspects of late steps in the replication of HBV, but appear to lack certain cellular components that play a pivotal role during early steps of the viral life cycle.