Dieter Glebe

Pre-S1 Antigen-Dependent Infection of Tupaia Hepatocyte Cultures with Human Hepatitis B Virus

ABSTRACT The susceptibility of the tree shrew Tupaia belangeri to human hepatitis B virus (HBV) has been demonstrated both in vivo and in vitro. In this study, we show that purified HBV infects primary T. belangeri hepatocyte cultures in a very specific manner, as detected by HBV covalently closed circular DNA, mRNA, HBV e antigen, and HBsAg production. A monoclonal antibody (MAb), MA18/7, directed against the pre-S1 domain of the large HBs protein, which has been shown to neutralize infectivity of HBV for primary human hepatocytes, also blocked infection of primary Tupaia hepatocytes. MAbs against the pre-S2 domain of HBs inhibited infection only partially, whereas an S MAb and polyvalent anti-HBs antibodies neutralized infection completely. Thus, both pre-S1 and S antigens are necessary for infection in the tupaia. Using subviral particles, >70% of primary Tupaia hepatocytes are capable of specific binding of pre-S1-rich HBsAg, showing localization in distinct membrane areas. The data show that the early steps of HBV infection in Tupaia hepatocyte cultures are comparable to those in the human system.

Role of glycosaminoglycans for binding and infection of hepatitis B virus.

Many parts of the life cycle of hepatitis B virus (HBV) infection of hepatocytes have been unravelled, but the attachment and entry process leading to infection is largely unknown. Using primary Tupaia hepatocyte cultures as an in vitro infection system, we determined that HBV uses cell‐surface heparan sulfate proteoglycans as low‐affinity receptor, because HBV infection was inhibited by heparin (IC50: 5 μg ml−1) or other higher‐sulfated polymers, but not by lower‐sulfated glycosaminoglycans, such as chondroitin sulfate. Pretreatment of primary hepatocytes with heparinase decreased viral binding and inhibited HBV infection completely. Interestingly, after preS1‐dependent viral binding at 16°C to the cell surface, subsequent infection could still be inhibited by HBV preS1‐lipopeptides, but not by heparin any more, suggesting a shift of the virus to a high‐affinity receptor. In summary, we suggest following multistep attachment process: in vivo, HBV is initially trapped within the liver in the space of Dissé by heparan sulfate proteoglycans. Thereafter, HBV binds via its preS1 attachment site and the N‐terminal myristic acid to a yet unknown, high‐affinity receptor that confers uptake in a yet unknown compartment.

Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.

Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes

Significance Hepatitis B virus (HBV) is the prototype hepadnavirus; 40% of humans have current or past infection. In a global investigation of viral diversity in bats, we discovered three unique hepadnavirus species. The relatedness of these viruses to HBV suggests that bats might constitute ancestral sources of primate hepadnaviruses. Infection patterns in bats resembled human infection with HBV. After resurrection from bat tissues, pseudotyped viruses carrying surface proteins of one bat hepadnavirus could infect human liver cells. HBV vaccination is probably not protective against these viruses, but viral replication could be blocked by a reverse transcriptase inhibitor used as an anti-HBV drug in humans. The potential of bat hepadnaviruses to infect humans should be considered in programs aimed at eradicating HBV. The hepatitis B virus (HBV), family Hepadnaviridae, is one of most relevant human pathogens. HBV origins are enigmatic, and no zoonotic reservoirs are known. Here, we screened 3,080 specimens from 54 bat species representing 11 bat families for hepadnaviral DNA. Ten specimens (0.3%) from Panama and Gabon yielded unique hepadnaviruses in coancestral relation to HBV. Full genome sequencing allowed classification as three putative orthohepadnavirus species based on genome lengths (3,149–3,377 nt), presence of middle HBV surface and X-protein genes, and sequence distance criteria. Hepatic tropism in bats was shown by quantitative PCR and in situ hybridization. Infected livers showed histopathologic changes compatible with hepatitis. Human hepatocytes transfected with all three bat viruses cross-reacted with sera against the HBV core protein, concordant with the phylogenetic relatedness of these hepadnaviruses and HBV. One virus from Uroderma bilobatum, the tent-making bat, cross-reacted with monoclonal antibodies against the HBV antigenicity determining S domain. Up to 18.4% of bat sera contained antibodies against bat hepadnaviruses. Infectious clones were generated to study all three viruses in detail. Hepatitis D virus particles pseudotyped with surface proteins of U. bilobatum HBV, but neither of the other two viruses could infect primary human and Tupaia belangeri hepatocytes. Hepatocyte infection occurred through the human HBV receptor sodium taurocholate cotransporting polypeptide but could not be neutralized by sera from vaccinated humans. Antihepadnaviral treatment using an approved reverse transcriptase inhibitor blocked replication of all bat hepadnaviruses. Our data suggest that bats may have been ancestral sources of primate hepadnaviruses. The observed zoonotic potential might affect concepts aimed at eradicating HBV.

Combination of DNA Prime – Adenovirus Boost Immunization with Entecavir Elicits Sustained Control of Chronic Hepatitis B in the Woodchuck Model

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients.

Occult hepatitis B virus infection: detection and significance.

The Taormina Consensus Conference defined ‘occult hepatitis B virus (HBV) infection’ (OBI) as the ‘presence of HBV DNA in the liver of individuals testing HBsAg-negative with currently available assays’. Most occult is the so-called ‘window period’ after exposure before HBV DNA appears in the blood. We identified two blood donors whose donations tested HBsAg- and HBV DNA-negative, but transmitted HBV. Both subsequently developed HBsAg and acute hepatitis. However, such cases are not considered as true OBI. A true transient OBI remains HBsAg-negative during the entire course. One case of acute OBI showed a peak viremia of 15,000 IU/ml HBV DNA and sub-borderline HBsAg, suggesting a ratio of virions to subviral particles of 1:10, whereas ‘normal’ cases show at peak viremia a ratio of 1:3,000. Blood donors with OBI may transmit HBV. We studied 5 blood donors with OBI and 55 of their recipients. In 22 recipients, transmission was probable, but they remained healthy. However, in 3 recipients, who were immunosuppressed at the time of transfusion, fatal fulminant hepatitis B developed. The majority of anti-HBc-positive healthy individuals have HBV DNA in the liver which may start replication under severe immunosuppression. Nine such cases are described here. OBI or reactivated HBV infections often lead to selection of HBsAg escape mutations as we could show in 11 of 14 cases. Infection of vaccinated individuals favors development of OBI as we observed in 6 blood donors. HB vaccination may solve the problem of overt HBV infection but may favor OBI.

Deficiencies in the standardization and sensitivity of diagnostic tests for hepatitis B virus.

Summary.  The patterns of hepatitis B virus (HBV) markers described in textbooks apply to acute and chronic infection with wild‐type HBV. Deviations from these patterns occur in the very early phase, in low‐level (or occult) infection and under immunosuppression. Variability may originate from the virus, the host or the test kits. In order to obtain a reliable diagnosis under these conditions, tests for all three markers of HBV infection have to be applied: HBsAg, HBV DNA and anti‐HBc. All tests should be as sensitive as feasible, but even then occult infection may be missed. Reliable detection of occult or mutated HBV is particularly important in blood and organ donors and in patients before or with immunosuppression.

The Molecular Virology of Hepatitis B Virus

Hepatitis B virus (HBV) is one of the smallest enveloped DNA viruses and the prototype member of the family of Hepadnaviridae that causes acute and chronic infections of mammals (including human) and birds. HBV has evolved an extreme adaptation and dependency to differentiated hepatocytes of its host. Despite its very limited coding capacity with only four open-reading frames, HBV is able to evade the immune system of the host and persist lifelong within infected hepatocytes. During active replication, HBV produces enormous viral loads in the blood and a massive surplus of subviral surface antigen particles in the serum of infected patients without killing their hepatocytes. Together with the use of a reverse transcriptase during replication, it provides an enormous genetic flexibility for selection of viral mutants upon selective pressure, for example, by the immune system or antiviral therapy. In addition, viral wild-type and mutated genomes are stably archived in the nucleus of the infected hepatocyte in an episomal DNA form that provides independence from cellular replication or integration within the host genome. We are just beginning to understand the delicate molecular and cellular interactions during the HBV replicative cycle within infected hepatocytes, so further studies are urgently needed to provide a better basis for further diagnostic and therapeutic options.

Hepatitis B virus infection is dependent on cholesterol in the viral envelope

The viral and cellular determinants leading to binding and entry of hepatitis B virus (HBV) are still not fully understood. We found that HBV infection of primary hepatocyte cultures is dependent on the presence of cholesterol in the viral envelope. Extraction of cholesterol from HBV purified from plasma of HBV‐infected patients with methyl‐beta‐cyclodextrin (MβCD) leads to a strongly reduced level of infection. The cholesterol‐depleted virions showed higher buoyant density (1.23 versus 1.17 g ml−1), a smaller diameter (39 versus 48 nm), but maintained particle integrity, antigenicity and ability to bind to hepatocytes. Although addition of exogenous cholesterol and cholesterol analogues restored the physical appearance of cholesterol‐depleted virions, infectivity was only regained by cholesterol add‐back. Infectivity of HBV produced from cell culture in the presence of inhibitors of cholesterol‐synthesis is severely impaired. Interestingly, cholesterol extraction from cellular membranes, incubation with filipin and the protein tyrosine kinase inhibitor genistein showed no effect on HBV infection, excluding a role of lipid rafts for the infection process of HBV. In summary, presence of cholesterol within the viral envelope is not important for viral binding, but indispensable for the entry process of HBV and might be important for a later step in viral uptake, e.g. fusion in a yet unknown compartment.

Analysis of the Pre-S2 N- and O-Linked Glycans of the M Surface Protein from Human Hepatitis B Virus

The surface antigen of hepatitis B virus comprises a nested set of small (S), middle (M), and large (L) proteins, all of which are partially glycosylated in their S domains. The pre-S2 domain, present only in M and L proteins, is furtherN-glycosylated at Asn-4 exclusively in the M protein. Since the pre-S2 N-glycan appears to play a crucial role in the secretion of viral particles, the M protein may be considered as a potential target for antiviral therapy. For characterization of the pre-S2 glycosylation, pre-S2 (glyco)peptides were released from native, patient-derived hepatitis B virus subviral particles by tryptic digestion, separated from remaining particles, purified by reversed-phase high performance liquid chromatography, and identified by amino acid and N-terminal sequence analysis as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Pre-S2 N-glycans were characterized by anion exchange chromatography, methylation analysis, and on target sequential exoglycosidase digestions in combination with MALDI-TOF-MS, demonstrating the presence of partially sialylated diantennary complex-type oligosaccharides. In addition, the pre-S2 domain of M protein, but not that of L protein, was found to be partially O-glycosylated by a Gal(β1–3)GalNAcα-, Neu5Ac(α2–3)Gal(β1–3)GalNAcα-, or GalNAcα-residue. The respective O-glycosylation site was assigned to Thr-37 by digestion with carboxypeptidases in combination with MALDI-TOF-MS and by quadrupole time-of-flight electrospray mass spectrometry. Analytical data further revealed that about 90% of M protein is N-terminally acetylated.