Initial sites of hepadnavirus integration into host genome in human hepatocytes and in the woodchuck model of hepatitis B-associated hepatocellular carcinoma.

Hepatitis B virus (HBV) and the closely related woodchuck hepatitis virus (WHV) are potent carcinogens that trigger development of primary hepatocellular carcinoma (HCC). The initial sites of hepadnavirus-host genome integration, their diversity and kinetics of formation can be central to virus persistence and the initiation and progression of HCC. To recognize the nature of the very early virus-host interactions, we explored de novo infection of human hepatocyte-like HepaRG cells with authentic HBV and naive woodchucks with WHV. HepaRG were analyzed from several minutes post exposure to HBV onwards, whereas woodchuck liver biopsies at 1 or 3 h and 6 weeks post infection with WHV. Inverse PCR and clonal sequencing of the amplicons were applied to identify virus-host genomic junctions. HBV and WHV DNA and their replication intermediates became detectable in one hour after virus exposure. Concomitantly, HBV DNA integration into various host genes was detected. Notably, junctions of HBV X gene with retrotransposon sequences, such as LINE1 and LINE2, became prominent shortly after infection. In woodchucks, insertion of WHV X and preS sequences into host genome was evident at 1 and 3 h post infection (h.p.i.), confirming that hepadnavirus under natural conditions integrates into hepatocyte DNA soon after invasion. The HBV and WHV X gene enhancer II/core promotor sequence most often formed initial junctions with host DNA. Moreover, multiple virus-virus DNA fusions appeared from 1 h.p.i. onwards in both infected hepatocytes and woodchuck livers. In summary, HBV DNA integrates almost immediately after infection with a variety of host's sequences, among which tandemly repeating non-coding DNAs are common. This study revealed that HBV can engage mobile genetic elements from the beginning of infection to induce pro-oncogenic perturbations throughout the host genome. Such swift virus insertion was also evident in natural hepadnaviral infection in woodchucks.

Hepatitis C virus upregulates B-cell receptor signaling: a novel mechanism for HCV-associated B-cell lymphoproliferative disorders.

B-cell receptor (BCR) signaling is essential for the development of B cells and has a critical role in B-cell neoplasia. Increasing evidence indicates an association between chronic hepatitis C virus (HCV) infection and B-cell lymphoma, however, the mechanisms by which HCV causes B-cell lymphoproliferative disorder are still unclear. Herein, we demonstrate the expression of HCV viral proteins in B cells of HCV-infected patients and show that HCV upregulates BCR signaling in human primary B cells. HCV nonstructural protein NS3/4A interacts with CHK2 and downregulates its activity, modulating HuR posttranscriptional regulation of a network of target mRNAs associated with B-cell lymphoproliferative disorders. Interestingly, the BCR signaling pathway was found to have the largest number of transcripts with increased association with HuR and was upregulated by NS3/4A. Our study reveals a previously unidentified role of NS3/4A in regulation of host BCR signaling during HCV infection, contributing to a better understanding of the molecular mechanisms underlying HCV-associated B-cell lymphoproliferative disorders.

Rare occurrence of occult hepatitis C virus in apparently uninfected injecting drug users: a two-centre, masked, case-control study.

Occult hepatitis C virus (HCV) is a phenomenon where serum HCV RNA is not detected by sensitive commercial assays, but viral RNA is detected by ultrasensitive techniques. Occult HCV infection has not previously been studied in highly exposed, but apparently uninfected (EU) individuals. Two studies examining occult infection in EU subjects were undertaken - an initial two-centre, masked, case-control study based on cross-sectional samples (n = 35 subjects) and a single-centre confirmatory study based on longitudinal samples (n = 32 subjects). Plasma and peripheral blood mononuclear cells were tested for HCV RNA using an ultrasensitive nested polymerase chain reaction assays. Two EU subjects in the first study (10%) and one in the second study (3%) were found to have consistently detectable HCV RNA. Occult HCV infection occurs in high-risk, apparently uninfected subjects.

Hepatitis C virus persistence after sustained virological response to antiviral therapy in patients with or without past exposure to hepatitis B virus.

Hepatitis C virus (HCV) and hepatitis B virus (HBV) frequently coinfect and persist long after clinical resolution. We assessed the incidence of low-level (occult) HCV infection (OCI) after sustained virological response (SVR) to standard anti-HCV therapy in individuals with or without past exposure to HBV to recognize whether HBV could influence the prevalence of OCI, HCV level and hepatic histology. Plasma and peripheral blood mononuclear cells (PBMC) were collected from 24 individuals at 6- to 12-month intervals for up to 72 months after SVR. Liver histology was available for nine patients. HCV and HBV genomes were detected with sensitivity <10 genome copies/mL. In individuals without HBV exposure (n = 15), comprehensive analyses of sequential plasma and PBMC samples revealed HCV RNA in all 15 cases (75% plasma and 61% PBMC). In the group with HBV exposure (n = 9), evidenced by circulating anti-HBc and/or HBV DNA detection by a highly sensitive assay, HCV RNA was identified in all cases (83% plasma and 59% PBMC), at levels similar to those in HBV nonexposed individuals. In both groups of patients, most liver biopsies included those reactive for viral genomes displayed low-grade inflammation (8 of 9) and fibrosis (7 of 9). Sequence polymorphisms at the 5`-UTR between PBMC and liver or plasma, as well as circulating HCV virion-like particles, were observed in patients with or without HBV exposure. In conclusion, the prevalence of OCI after SVR is comparable in individuals with or without past exposure to HBV. HCV loads and liver alterations in OCI appear to be unaffected by low-level HBV DNA carriage.

Molecular characterization of intrahepatic and extrahepatic hepatitis B virus (HBV) reservoirs in patients on suppressive antiviral therapy.

The hepatitis B virus (HBV) replicates via an error-prone reverse transcriptase generating potential drug-resistant quasispecies. The degree of HBV variability in liver vs peripheral blood mononuclear cells (PBMC) in patients on long-term suppressive antivirals is unclear. We characterized HBV replication, drug resistance and molecular diversity in patients with plasma HBV DNA undetectable by clinical assays. Explant liver (n=9), PBMC (n=6) and plasma (n=7) from nine such patients undergoing liver transplantation were evaluated for HBV genomes by sensitive PCR/nucleic acid hybridization assay. Cases with HBV DNA in liver and PBMC were tested for covalently closed circular DNA (HBV cccDNA). HBV polymerase (P) amplicons were cloned, sequenced and both P and overlapping surface (S) gene sequences were analysed. HBV DNA was detected in 43% (3/7) of plasma, 100% (9/9) of liver and 83% (5/6) of PBMC samples. HBV cccDNA was detected in all liver and one PBMC sample. Four patients had a clinical diagnosis of resistance. HBV P gene sequencing revealed 100% wild type (wt) in plasma (2/2), 83% wt in PBMC (5/6) but livers of 3/9 (33%) contained wt and 6/9 (66%) carried resistance to lamivudine and/or adefovir. The translated S gene revealed no changes affecting HBV antigenicity. Sequences from livers with antiviral resistant mutants revealed greater interpatient quasispecies diversity. Despite apparent HBV suppression, the liver continues to support HBV replication and extrahepatic HBV can be detected. PBMC may be a sanctuary for wt virus during antiviral therapy, while the liver harbours more drug-resistant viruses. Drug resistance correlates with intrahepatic viral diversity.

Chronic hepatitis C and persistent occult hepatitis C virus infection are characterized by distinct immune cell cytokine expression profiles.

Hepatitis C virus (HCV) replicates in immune cells in both chronic hepatitis C (CHC) and occult HCV infection, but the extent of virus replication in this compartment in these opposing infection forms varies greatly. It was unknown whether this could be linked to HCV genotype or to differences in host gene expression shaping the immune response, and whether HCV replication in immune cells is sensitive to endogenous antiviral cytokines. In this study, we uncovered that significantly greater HCV load in peripheral blood mononuclear cells (PBMC), but not in plasma, coincided with HCV genotypes 2 and 3 in CHC, but with genotype 1 in residual occult infection after clinical resolution of hepatitis C. Moreover, PBMC from individuals with occult infection transcribed significantly greater levels of IFN-alpha, IFN-gamma and TNF-alpha, but less interleukin (IL)-10 than those from CHC. In CHC, PBMC with low HCV load expressed significantly more IFN-gamma but less IL-12 than did cells with high virus content. In occult infection, HCV RNA detection in PBMC was associated with much lower IFN-alpha and IL-12 expression. Further, HCV replication in T lymphocytes could be completely eliminated by activation of endogenous IFN-gamma in CHC, but of IFN-alpha in occult infection. In conclusion, CHC and persistent occult HCV infection are characterized by clearly different profiles of antiviral cytokine response in circulating immune cells which are also different from those of healthy individuals. Higher expression of IL-10, combined with lower transcription of IFN-alpha, IFN-gamma and TNF-alpha, is associated with a more robust HCV replication in immune cells.

Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infection.

Detection of residual HCV in individuals with SVR after treatment of CHC can be significantly heightened by analyzing ex vivo mitogen-activated T and B lymphocytes and applying sensitive nucleic acid amplification assays. However, it remained unknown if synergistic activation of lymphocytes and monocytes would further augment HCV detection, if viral replication becomes universally upregulated in treated cells, and if examining sequential sera and lymphoid cells would improve detection of occult infection. Using paired sera and lymphoid cells collected 1 year apart from 17 individuals with normal liver enzymes for up to 72 months after SVR, it was found that simultaneous activation of lymphocytes and monocytes enhanced identification of silent HCV infection and revealed that in some cases monocytes were the principal immune cell type where HCV persisted. Testing of serial samples further increased detection of occult infection. Ultimately, by combining the above two approaches, all individuals with SVR were found to be silent carriers of HCV. Clonal sequencing revealed HCV variations in sera and lymphoid cells and evolution of viral genomes confirming ongoing virus replication. Surprisingly, similar to those with CHC, naive lymphoid cells from some individuals carried approximately 10(3) HCV copies/microg total RNA. HCV loads in naive lymphoid cells predetermined the outcome of ex vivo stimulation with respect to upregulation or inhibition of HCV replication. HCV RNA levels in occult infection were inversely proportional to the expression of IFNalpha and IFN-inducible MxA, but not to IFNgamma or tumour necrosis factor alpha in naive and mitogen-treated lymphoid cells.

Comparison of biological activity of recombinant woodchuck interferon gamma and tumor necrosis factor alpha produced in baculovirus and Escherichia coli expression systems.

The full-length cDNAs of recombinant woodchuck interferon gamma (rwIFN gamma) and woodchuck tumor necrosis factor alpha (rwTNF alpha) were cloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The recombinant proteins secreted by the insect cells, bac-rwIFN gamma and bac-rwTNF alpha, were found to be functionally competent. Their biological activities were compared to those of rwIFN gamma and rwTNF alpha produced in the Escherichia coli (E. coli) expression system. The bac-rwIFN gamma demonstrated a 4.5-fold greater protective activity against encephalomyocarditis virus-induced cytolysis of woodchuck hepatocytes and that of class I MHC antigen presentation on the hepatocytes than rwIFN gamma derived from E. coli. The bac-rwTNF alpha was cytotoxic towards murine fibroblasts and able to upregulate class I MHC antigen display and these effects were about 18-fold greater than those triggered by rwTNF alpha from E. coli at a comparable protein level. In addition, the antiviral activity of bac-rwIFN gamma was inhibited by anti-wIFN gamma antibodies and the cytotoxicity of bac-rwTNF alpha neutralized by cross-reactive antibodies to murine TNF alpha. The study showed that the expression of rwIFN gamma and rwTNF alpha in the baculovirus system generated biologically active cytokines whose potency was considerably greater than those produced in E. coli.

Augmented hepatic interferon gamma expression and T-cell influx characterize acute hepatitis progressing to recovery and residual lifelong virus persistence in experimental adult woodchuck hepatitis virus infection.

Woodchucks infected with woodchuck hepatitis virus (WHV) have profiles of liver disease and age-dependent rates of progression to chronic hepatitis (CH) comparable with those seen in human hepatitis B. The mechanism of recovery from acute hepadnaviral infection or its evolution to chronicity remains unknown, although the liver immune responses are expected to play an important role. To determine the dynamics of intrahepatic cytokine expression and T-cell involvement, and to assess their value in predicting the outcome of acute hepatitis (AH) in the adult onset of WHV infection, we evaluated liver transcription of interferon gamma (IFN-gamma); tumor necrosis factor alpha (TNF-alpha); interleukins (IL)-2, -4, and -6; and the T-cell influx in relation to disease histologic severity and virus load in serial liver biopsies collected during the life span of experimentally infected woodchucks. Our results show that recovery from viral AH in adulthood is preceded by a significantly greater hepatic expression of IFN-gamma and CD3, an increased TNF-alpha transcription, lower hepatic WHV load, and a greater degree of liver inflammation than those in acute infection with CH outcome. Furthermore, we have learned that the elevated IFN-gamma, TNF-alpha, and CD3 expression in the liver endures for years not only in CH, but also, although to a lesser extent, in apparently completely resolved infection. This is consistent with our previous findings that residual WHV replication and remnant liver inflammation continue for life after recovery from AH. This study indicates that antiviral cytokines, in particular IFN-gamma, may play a central role in the long-term control of occult hepadnavirus persistence in the liver.

In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus.

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.

Occult persistence and lymphotropism of hepadnaviral infection: insights from the woodchuck viral hepatitis model.

Hepatitis B virus (HBV) is a major human pathogen that causes chronic infection and life-threatening liver diseases in millions of individuals. While pathological and epidemiological consequences of clinically evident HBV infections are well recognized, there is no similar knowledge on an asymptomatic, silently progressing virus persistence. Contrary to previous opinion, current evidence indicates that a serologically undetectable (occult) HBV carriage is a common outcome of recovery from symptomatic illness and that scanty amounts of the virus are carried by apparently healthy individuals for years after resolution of hepatitis B despite the presence of presumably protective antiviral antibodies. Recent studies on this silent form of hepadnavirus carriage in an experimental woodchuck hepatitis virus (WHV) infection, which is considered to be the closest natural model of HBV disease, revealed that the life-long occult persistence of traces of pathogenic virus is an invariable consequence of recovery after hepadnaviral invasion and that this state always co-exists with a steady low-rate virus replication in both the liver and the lymphatic system. Importantly, this serologically concealed infection can be accompanied by development of hepatocellular carcinoma in convalescent animals and is transmittable from mothers to offspring as an asymptomatic, indefinitely long infection which involves the lymphatic system but not always the liver. This review focuses on the features of hepadnavirus occult persistence and its lymphotropism, and on what is currently understood about the contribution of the lymphatic system in maintaining hepadnavirus carriage based on insights provided by analysis of the woodchuck-WHV experimental system.

Posttranscriptional inhibition of class I major histocompatibility complex presentation on hepatocytes and lymphoid cells in chronic woodchuck hepatitis virus infection.

Woodchuck hepatitis virus (WHV), similar to human hepatitis B virus, causes acute liver inflammation that can progress to chronic hepatitis and hepatocellular carcinoma. WHV also invades cells of the host lymphatic system, where it persists for life. We report here that acute and chronic hepadnavirus hepatitis is characterized by a profound difference in the expression of class I major histocompatibility complex (MHC) molecules on the surface of infected hepatocytes and, notably, lymphoid cells. While acute WHV infection is accompanied by the enhanced hepatocyte surface presentation of class I MHC antigen and upregulated transcription of the relevant hepatic genes, inhibition of class I antigen display on liver cells is a uniform hallmark of chronic WHV infection. This inhibition in chronic hepatitis occurs despite augmented (as in acute infection) expression of hepatic genes for class I MHC heavy chain, beta(2)-microglobulin, and transporters associated with antigen processing (TAP1 and TAP2). Further, the class I antigen inhibition is not related to the histological severity of hepatocellular injury, the extent of lymphocytic infiltrations, the level of intrahepatic gamma interferon induction, or the hepatic WHV load. Importantly, the antigen expression is also inhibited on organ lymphoid cells of chronically infected hosts. The results obtained in this study demonstrate that the defective presentation of class I MHC molecules on cells supporting persistent WHV replication is due to viral posttranscriptional interference. This event may diminish the susceptibility of infected hepatocytes to virus-specific T-cell-mediated elimination, hinder virus clearance, and deregulate the class I MHC-dependent functions of the host immune system. This multifarious effect could be critical for perpetuation of liver damage and evasion of the antiviral immunological surveillance in chronic infection and therefore could be supportive of hepadnavirus persistence.

Perforin and Fas/Fas ligand-mediated cytotoxicity in acute and chronic woodchuck viral hepatitis.

The Fas ligand (FasL)/Fas and the perforin-granzyme cytotoxic pathways presumably play a central role in the development of hepatocellular injury in viral hepatitis. To recognize the potential contribution of FasL and perforin-based cell killing in hepadnaviral infection, we adopted a cytotoxic assay using murine Fas+ P815 and human Fas- K562 cells as targets. Freshly isolated peripheral blood mononuclear cells (PBMC) from woodchucks with newly acquired woodchuck hepatitis virus (WHV) infection (n = 6), with chronic WHV hepatitis (n = 9), and from healthy animals (n = 11) were used as effector cells. We have found that woodchuck lymphoid cells kill cell targets via both the FasL/Fas and the perforin death pathways. The contribution of Fas-dependent cytolysis was ascertained in blocking experiments with anti-Fas antibody and by incubation of PBMC with cyclohexamide to prevent de novo synthesis of FasL. The involvement of the perforin pathway was confirmed by treatment of K562 cells with colchicine to inhibit the microtubule-dependent perforin release. Comparative analysis showed that peripheral lymphoid cells from acute WHV hepatitis, but not those from chronic WHV infection, are more cytotoxic and that this increase seems to be entirely due to activation of perforin-mediated killing. The data indicate that acute infection in woodchucks is associated with the augmented capacity of lymphoid cells to elicit perforin-dependent killing, but in chronic infection, independent of the severity of liver disease and duration of chronicity, these cells have the same or lower cytotoxic potential as PBMC from healthy controls. These findings suggest a role for non-specific cellular immunity, presumably natural killer (NK) cells, in the control of early WHV infection and in the progression of chronic hepatitis.

Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis.

Mother-to-child transmission is an important route for hepatitis B virus (HBV) dissemination. It has been established that HBV traces persist for years after complete clinical recovery from hepatitis B. Similarly, resolution of hepatitis caused by HBV-related woodchuck hepatitis virus (WHV) is followed by occult lifelong carriage of pathogenic virus. In this study, we documented that WHV persisting after termination of acute hepatitis is transmittable to newborns as an asymptomatic long-term infection. All 11 offspring from 4 dams studied carried transcriptionally active WHV genomes for 3.5 years after birth without immunovirological markers of infection. WHV genomes and mRNA were detected both in the liver and lymphoid tissue in the majority of offspring; WHV covalently closed circular DNA was detected in some samples. In 4 offspring, however, the virus was restricted to the lymphatic system. In the circulation, WHV DNA-reactive particles were DNase resistant and of comparable size and density to complete virions. Importantly, the virus in offspring with or without hepatic WHV DNA expression was infectious to WHV-naive woodchucks. Finally, offspring challenged with WHV were not protected against reinfection. These findings show that mothers with occult hepadnaviral carriage transmit pathogenic virus to their offspring, inducing a persistent infection invariably within the lymphatic system but not always in the liver.

Occult lifelong persistence of infectious hepadnavirus and residual liver inflammation in woodchucks convalescent from acute viral hepatitis.

Traces of hepatitis B virus (HBV) genome can persist for years following recovery from hepatitis B. To determine overall duration, molecular characteristics, and pathological implications of this serologically undetectable form of hepadnaviral carriage, we have analyzed the expression of transcriptionally active virus genomes, their infectivity, and examined liver alterations during the natural lifespan of woodchucks convalescent from acute infection with HBV- related woodchuck hepatitis virus (WHV). In this study, we document lifelong persistence of scanty amounts of replicating virus both in the liver and lymphatic system after spontaneous resolution of an episode of experimental hepadnaviral hepatitis. Antibodies to virus nucleocapsid (core) were found to be the most reliable immunovirological marker coexisting with occult infection. In the majority of convalescent woodchucks, serial liver biopsies showed protracted minimal to mild necroinflammation with periods of normal morphology; however, hepatocellular carcinoma (HCC) ultimately developed in 2 of 9 animals studied. Inocula derived from lymphoid cells of convalescent animals induced classical acute hepatitis in virus-naive woodchucks that progressed to chronic hepatitis and HCC in 1 of the animals, demonstrating infectivity and pathogenic competence of the carried virus. Our results reveal that low levels of infectious WHV and residual hepatic inflammation usually continue for life after resolution of hepatitis and that this recovery does not avert HCC development. They also demonstrate that, in addition to the liver, the lymphatic system is the site of the occult lifelong maintenance of replicating hepadnavirus.

Complement-mediated cytotoxicity and inhibition of ligand binding to hepatocytes by woodchuck hepatitis virus-induced autoantibodies to asialoglycoprotein receptor.

Hepadnavirus invasion in woodchucks has been identified as a potent inducer of autoantibodies against asialoglycoprotein receptor (anti-ASGPR), a molecule essentially unique to hepatocytes that mediate clearance of desialylated serum proteins. We evaluated the possible pathogenetic importance of anti-ASGPR triggered by woodchuck hepatitis virus (WHV), using anti-ASGPR-reactive serum immunoglobulins (Igs) from five animals with different stages of WHV hepatitis or self-limited WHV infection and isolated woodchuck hepatocytes or HepG2 cells as targets. The results revealed that WHV-induced anti-ASGPR can specifically inhibit asialoglycoprotein recognition by both homologous and heterologous liver cells, as tested in an asialofetuin (ASFN)-binding radioassay. However, the extent of the interference significantly varied (from 85% inhibition to none) for anti-ASGPR with similar titer from different animals, indicating a high degree of heterogeneity in the ASGPR epitope specificity and in the potential biological effects of these autoantibodies. The WHV-triggered anti-ASGPR also induced complement-mediated hepatocytolysis in a microculture tetrazolium (MTT) assay, which ranged from 8.9% +/- 0.3% to 33.6% +/- 3.6% (mean +/- SD) for different animals and target cell numbers. This cytopathic effect was strictly ASGPR-specific, complement-dependent, and was not related to the anti-ASGPR ability to inhibit ligand-hepatocyte binding. Our findings indicate that among pathways by which anti-ASGPR autoimmunity could cause liver damage, hepadnavirus-induced anti-ASGPR might impair hepatocytes by both disrupting clearance of desialylated proteins and activation of the complement-mediated cytolysis. These cytopathic effects might contribute to the pathogenesis, aggravate severity, and prolong recovery from liver injury in viral hepatitis.

Virus-induced anti-asialoglycoprotein receptor autoimmunity in experimental hepadnaviral hepatitis.

The relationship between hepatitis virus invasion and emergence of liver-specific autoantibodies against asialoglycoprotein receptor (anti-ASGPR) and the occurrence patterns, prognostic value, and specificity of these autoantibodies toward polypeptides of host ASGPR were investigated in experimental viral hepatitis in the woodchuck system. Sequential sera (n = 231) obtained before and after inoculation with woodchuck hepatitis virus (WHV) from animals which resolved acute infection (n = 7) or developed chronic hepatitis (n = 6) were tested for anti-ASGPR using radio and enzyme-immunodetection assays. In addition, the outcome of WHV hepatitis was analyzed in 30 other woodchucks whose preinoculation sera were tested for anti-ASGPR. The receptor subunit specificity of virus-induced anti-ASGPR was determined by Western blotting and compared with that of anti-ASGPR raised in woodchucks challenged with a heterologous (rabbit) receptor. The results revealed that WHV infection triggered anti-ASGPR in all except one of the initially autoantibody nonreactive animals (eight of nine; 89.9%). Once induced, anti-ASGPR were detectable throughout the entire follow-up independent of histological severity of liver damage or the outcome of hepatitis. In healthy WHV-naive woodchucks, anti-ASGPR occurred at low titers in approximately one third of the animals. Importantly, woodchucks reactive for anti-ASGPR before WHV inoculation developed chronic hepatitis with a significantly greater frequency (55.5%) than those autoantibody negative (15.6%; P < .05). In contrast to anti-ASGPR elicited by immunization with a heterologous receptor, which initially recognized only the ASGPR 40-kd polypeptide, anti-ASGPR emerging after virus invasion reacted with both the ASGPR 40- and 47-kd subunits from the moment of their appearance. This study provides the first direct evidence that hepatitis virus in the natural host triggers autoantibodies against a unique hepatocyte antigen and shows that anti-ASGPR autoimmunity existing before virus infection is associated with a high rate of progression to chronic disease in experimental hepadnaviral hepatitis.

Composition, antigenic properties and hepatocyte surface expression of the woodchuck asialoglycoprotein receptor.

We have purified woodchuck hepatic asialoglycoprotein receptor (ASGPR) by ligand affinity chromatography and have identified it as a heterooligomeric complex comprised of two subunits with molecular masses of 40 and 47 kD, designated as woodchuck hepatic lectin 1 and 2 (WHL1 and WHL2), respectively. With the help of antisera generated against the soluble, bioactive woodchuck and rabbit ASGPRs and anti-subunit monospecific antibodies, distinct antigenic specificity of each of the ASGPR polypeptide subunits and interspecies immunologic cross-reactivity of the receptor polypeptides displaying comparable molecular masses were documented. In contrast to the purified woodchuck receptor, WHL2 antigenic reactivity was not identifiable in woodchuck hepatocyte plasma membranes unless the intact membranes were exposed to an asialylated ligand or a soluble membrane fraction was incubated with anti-receptor antibody. These findings imply that both WHL1 and WHL2 are expressed on the hepatocyte surface and contribute to ligand binding, since antibody specific to either subunit blocks ligand attachment. Our results also indicate that ligand binding modifies antigenic properties of the membrane expressed ASGPR.

Protease-activated lymphoid cell and hepatocyte recognition site in the preS1 domain of the large woodchuck hepatitis virus envelope protein.

A site capable of strictly host- and cell type-specific recognition was identified in the preS1 domain of woodchuck hepatitis virus (WHV) through the use of antipeptide antisera generated against the extreme N-terminal fragment of the large virus envelope protein. The crucial determinant of this binding site was mapped to amino acids 10-13. Although a synthetic analogue of the site was highly immunogenic, natural WHV envelope did not display the site activity unless it was modified by proteolysis or acidic pH treatment, indicating an internal location of the determinant in viral envelope. Synthetic peptides encompassing the sequence of this site bound woodchuck lymphoid cells and hepatocytes in a species-restricted manner which followed characteristics of a specific ligand-receptor interaction, although their ability to interact with lymphoid cells was considerably greater than that for hepatocytes. In WHV-infected animals, a natural antibody to the identified cryptic cell-binding site arose independently of that directed against epitopes of unmodified virus envelope and its appearance constituted the earliest immunovirological indicator of virus invasion. Our results demonstrated that the preS1 domain of the large WHV envelope protein is endowed with the species- and cell type-specific recognition site which is protected against antibody surveillance by the natural tertiary structure of the protein and we suggest that proteolytic cleavage is required to induce the binding activity.

Antibody-directed complement-mediated cytotoxicity to hepatocytes from patients with chronic hepatitis B.

The susceptibility of hepatocytes from patients with chronic hepatitis B to complement-dependent cytotoxicity mediated by heterologous antibodies to hepatitis B virus core (anti-HBc) and surface (anti-HBs) antigens and to hepatic asialoglycoprotein receptor was examined using a microcytotoxicity assay. The anti-HBc-induced cytotoxicity was found to be markedly enhanced against hepatocytes isolated from patients with chronic active hepatitis (72.6 +/- 9.5% (mean +/- s.e.m.); n = 6) over that against hepatocytes from individuals with chronic persistent hepatitis or inactive liver cirrhosis (40.6 +/- 18.6%; n = 4) (P = 0.019). Overall, values of the anti-HBc-directed cytotoxicity were higher in patients positive for HBcAg in hepatocytes and seropositive for hepatitis B virus e antigen (HBeAg). Hepatocytotoxicity was also exerted by anti-HBs and anti-asialoglycoprotein receptor antibodies in the presence of complement, but it was not seemingly related to disease activity. These results indicate that hepatitis B virus core and surface antigens and asialoglycoprotein receptor at the hepatocyte surface can be recognized by antibodies, and raise the possibility that complement-dependent cytolysis may contribute to the injury of hepatitis B virus-infected hepatocytes. The data also suggest that liver cells of patients with severe chronic hepatitis might be more susceptible to anti-HBc antibody-directed complement-mediated cytotoxicity than those with inactive liver histology.