In vitro response to HBsAg of peripheral blood lymphocytes from recipients of hepatitis B vaccine

Lymphocytes isolated from recipients of hepatitis B vaccine were studied for their immune response to HBsAg in vitro. Peripheral blood mononuclear cells (PBMs) from 70 to 80% of 40 vaccinees yielded proliferative indices larger than 2 after 5 to 7 days incubation with HBsAg. This in vitro proliferative response could be augmented by incubating the cells with HBsAg and supernatants of activated T cells for 2 weeks or longer. After 7 to 10 days, in vitro stimulation with antigen, PBMs (1 X 10(6] could yield 5 to 15 HBsAg-specific antibody-secreting plaque-forming cells. The antibody to HBsAg produced in vitro was greatly increased in cultures that contained antigen-specific B cells enriched by panning with HBsAg-coated plates and a T cell growth factor-dependent, HBsAg-specific autologous T cell line. The results indicate that HBsAg-specific B and T cells are present, although at low frequencies, in the circulation of hepatitis B vaccinees.     

Modulation of the immunological response to hepatitis B virus by antibodies

Antibodies to HBsAg of IgG class enhanced the helper activity of a human T cell clone to promote the in vitro synthesis of immunoglobulins by autologous B lymphocytes. Using two different assay systems, the effect of antigen-specific antibodies on the helper function of a HBsAg-reactive T cell clone was studied. The monoclonal antibody to HBsAg A5C3 (IgG) increased significantly the T cell-dependent production of immunoglobulins by Staphyloccocus aureus-stimulated autologous B lymphocytes. Furthermore, the results obtained with a different type of assay showed that A5C3 also increased the synthesis of antibody to HBsAg by the autologous B cells in the presence of HBsAg and the helper T cell clone. On the other hand, when the monoclonal antibody to HBsAg of IgM class, H5D3 or the F(ab')2 fragment of A5C3 were tested, no significant enhancement of the helper activity of the T cell clone was observed. Experiments performed in mice showed that the in vivo antibody to HBsAg response to low concentrations of HBsAg was significantly enhanced by mixing this antigen with monoclonal antibody to HBsAg of IgG class. No effect was observed when a monoclonal antibody to HBsAg of IgM class was used to prepare the immune-complexed immunogen. The results presented here suggest that antibodies play a critical role in their own production through regulating the activity of helper T cells. This phenomenon might contribute to the increased antibody synthesis of in vivo secondary immune responses and could be of use in designing more efficient vaccine programs in man.     

In vitro synthesis and immunoregulation of antibody to hepatitis B surface antigen in man

Abstract Peripheral blood mononuclear cells (PBMC) from 16 chronic hepatitis B surface antigen (HBsAg) carriers and from 10 immune individuals (anti-HBs and/or anti-HBc core antigen positive) were studied for their ability to synthesize antibody to hepatitis B viral antigens in vitro . Pokeweed mitogen (PWM)-stimulated B lymphocytes from carriers, synthesized polyclonal IgG and IgM normally but did not synthesize detectable antibody to HBsAg (anti-HBs) even in the presence of T lymphocyte help and the absence of T lymphocyte suppression from immune individuals. In contrast, B lymphocytes from 80% of immune individuals synthesized anti-HBs in vitro. In cell-mixing experiments, T lymphocytes from carriers were found to provide normal helper function for immunoglobulin and anti-HBs production by B lymphocytes from immune individuals. In addition, the degree of suppressor T lymphocyte activity of chronic carriers was not sufficient to explain the lack of anti-HBs production.
The effect of purified HBsAg on anti-HBs synthesis by PBMC from immune individuals was determined. Incubating PBMC for periods ranging from 10 min to 10 days in the presence of concentrations of HBsAg varying from 10 pg/ml to 10 μg/ml had no effect on the synthesis of anti-HBs by PBMC. These results suggest that chronic HBsAg carriers lack circulating B lymphocytes capable of producing anti-HBs and that this cannot be explained by the presence of large amounts of circulating HBsAg.     

Hepatitis B antigen-associated periarteritis nodosa in patients undergoing long-term hemodialysis

Periarteritis nodosa was observed in three of 266 persistent hepatitis B antigen (HBsAg) carriers undergoing long-term hemodialysis; no cases of necrotizing vasculitis occurred among 384 other patients undergoing dialysis having either no or transient antigenemia. Circulating e antigen, but no e antibody, was found in two of these three patients. The serum level of the third component of complement (C3) was normal in two patients and low in the third. Circulating immune complexes were demonstrated in all three patients, using polyethylene-glycol (PEG) precipitation, PEG-C4, and solid phase C1q tests. HBsAg and anti-hepatitis B antibody (HBsAb) were identified in the PEG precipitates using radioimmunoassay and electron microscopy technics. Direct immunofluorescence performed on a muscle biopsy specimen from one patient was positive for HBsAg, but not for immunoglobulin G (IgG), immunoglobulin M (IgM), C3 or C1q. These data support the hypothesis that circulating immune complexes involving HBsAg may be involved in the pathogenesis of periarteritis nodosa.     

Ultrastructural studies of fibroblasts transfected with hepatitis B virus DNA

Cultured 3T3 mouse fibroblasts transfected with cloned hepatitis B virus genome and DNA coding for methotrexate-resistant dihydrofolate reductase, produce and secrete significant amounts of hepatitis B surface antigen (HBsAg). Ultrastructural morphometry revealed that fibroblasts transfected with hepatitis B virus DNA contained significantly more lysosomes than did fibroblasts transfected with the gene coding for methotrexate resistance or normal fibroblasts. Although abundant HBsAg was found in the cytoplasm of transfected fibroblasts by immunologic methods, HBsAg particles were not detected by electron microscopy. Immunoelectron microscopy localized HBsAg to the nuclear envelope, rough endoplasmic reticulum, and endoplasmic cisternae. These findings suggest that the transfected cells produce mainly nonparticulate HBsAg or that they have a defect in intracisternal packaging of HBsAg into particles.     

Reduced hepatitis B virus surface antigen-specific Th1 helper cell frequency of chronic HBV carriers is associated with a failure to produce antigen-specific antibodies in the trimera mouse

In chronic hepatitis B virus (HBV) infection weak antiviral immune responses are associated with viral persistence. We studied possible immune deficits underlying the lack of serum antibodies of such patients against the HBV surface antigen (HBsAg) in a novel human/mouse chimeric model. A hepatitis B surface antigen (HBs) vaccination of Balb/c mice engrafted with peripheral blood mononuclear cells (PBMC) of naturally HBV-immunized donors induced high frequencies of human HBsAg-specific B and T helper 1 (Th1) cells. These responses were associated with high serum anti-HBs antibody levels of the subclasses immunoglobulin G1 (IgG1) and IgG2 that are driven by interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In contrast, PBMC of chronic HBV carriers transplanted into the chimera failed to produce anti-HBs antibodies after vaccination with HBsAg and exhibited a deficit of antigen-specific Th1 cells. A possible influence of HBsAg or viremia was excluded by the lack of viral replication in such chimera. The observed T-cell defect was specific for HBsAg, as the B- and T-cell responses to tetanus toxoid (TT) were fully retained. Thus, our study shows that viral persistence in chronic HBV carriers is associated with an HBsAg-specific Th1 cell defect, which likely is responsible for the insufficient neutralizing anti-HBs-antibody response and is not reversed by HBs vaccination. Alternative approaches to induce HBs-specific Th1 cell responses might represent a future therapeutic option.     

Current and novel modalities for management of chronic hepatitis B infection

Over 296 million people are estimated to have chronic hepatitis B viral infection (CHB), and it poses unique challenges for elimination. CHB is the result of hepatitis B virus (HBV)-specific immune tolerance and the presence of covalently closed circular DNA as mini chromosome inside the nucleus and the integrated HBV. Serum hepatitis B core-related antigen is the best surrogate marker for intrahepatic covalently closed circular DNA. Functional HBV “cure” is the durable loss of hepatitis B surface antigen (HBsAg), with or without HBsAg seroconversion and undetectable serum HBV DNA after completing a course of treatment. The currently approved therapies are nucleos(t)ide analogues, interferon-alpha, and pegylated-interferon. With these therapies, functional cure can be achieved in < 10% of CHB patients. Any variation to HBV or the host immune system that disrupts the interaction between them can lead to reactivation of HBV. Novel therapies may allow efficient control of CHB. They include direct acting antivirals and immunomodulators. Reduction of the viral antigen load is a crucial factor for success of immune-based therapies. Immunomodulatory therapy may lead to modulation of the host immune system. It may enhance/restore innate immunity against HBV (as toll-like-receptors and cytosolic retinoic acid inducible gene I agonist). Others may induce adaptive immunity as checkpoint inhibitors, therapeutic HBV vaccines including protein (HBsAg/preS and hepatitis B core antigen), monoclonal or bispecific antibodies and genetically engineered T cells to generate chimeric antigen receptor-T or T-cell receptor-T cells and HBV-specific T cells to restore T cell function to efficiently clear HBV. Combined therapy may successfully overcome immune tolerance and lead to HBV control and cure. Immunotherapeutic approaches carry the risk of overshooting immune responses causing uncontrolled liver damage. The safety of any new curative therapies should be measured in relation to the excellent safety of currently approved nucleos(t)ide analogues. Development of novel antiviral and immune modulatory therapies should be associated with new diagnostic assays used to evaluate the effectiveness or to predict response.     

Hepatitis B surface antigen specific cytotoxic T lymphocyte activity in hepatitis B virus infection

Cytotoxic T lymphocyte (CTL) activity against HBs antigen (HBsAg)-coated autologous mononuclear cells as target cells was examined in hepatitis B virus infection. Target cells were prepared by coincubation of mononuclear cells with purified HBsAg in 0.5 mM CrCl3. The distribution and uniformity amounts of HBsAg on target cells was shown by immune fluorescence and by analyzing the fluorescence intensity with a fluorescent activated cell sorter. CTL activity was detected in 7 of 9 patients with acute hepatitis type B, in 4 of 11 chronic active hepatitis type B, in none of 8 healthy HBsAg carriers, and in none of 22 healthy volunteers. The antigen specificity of the cytotoxicity was confirmed by a blocking experiment with purified HBsAg and by cold target inhibition with HBsAg or bovine serum albumin (irrelevant antigen) coupled cold target cells. CTL lysed HBsAg coupled allogeneic target cells that shared HLA-A or B locus antigens. This finding suggests that HLA restriction may be involved in the killing mechanism. This HBsAg specific CTL clone may participate in the immunopathogenesis of hepatitis B virus infection.     

Circulating immune complexes in acute viral hepatitis, chronic active hepatitis, and periarteritis nodosa (author's transl)]

Circulating immune complexes were determined in patients with acute viral hepatitis, chronic active hepatitis and periarteriitis nodosa with the Raji cell technique. Circulating immune complexes were found in 11/18 HBsAg positive and HBsAg negative cases of acute viral hepatitis. In HBsAg positive chronic active hepatitis immune complexes were detectable in 42/43 cases but only in 1/27 HBsAg negative cases. Ten healthy HBsAg carriers demonstrated no detectable immune complexes. Using FITC conjugated antisera against HBs, HBc, and e antigen, immune complexes could not be found in any case of acute viral hepatitis or chronic active hepatitis. Elution of immune complexes from Raji demonstrated IgG, C'3 and a lack of HBsAg, HBc or e antigen. Immune complexes were present in 4/8 cases with periarteriitis nodosa. Viral components were detectable in one case.     

Hyperviscosity syndrome associated with an idiopathic monoclonal IgA-rheumatoid factor

We report the clinical features and results of serologic studies of a patient with an idiopathic hyperviscosity syndrome whose serum contained large amounts of immunoglobulin-anti-immunoglobulin immune complexes. The intermediate-sedimenting (i.e., between 7S and 19S) nature of the circulating immune complexes was demonstrated by sucrose density gradient ultracentrifugation and gel filtration analyses. By analytic ultracentrifugation, the predominant peak of intermediate-sedimenting protein was calculated to be ~14S; no high molecular weight protein aggregates (>19S) were apparent even when undiluted serum was examined. The immunoglobulin with rheumatoid factor activity and the immunoglobulin-antiimmunoglobulin immune complexes were isolated and characterized. The rheumatoid factor activity was attributed to a monomeric, monoclonal 7S IgA_k protein and its anti-immunoglobulin activity localized to the F(ab')₂ fragment. The immune complexes consisted of the monoclonal immunoglobulin A (IgA) protein and polyclonal immunoglobulin G (IgG). The immune complexes were dissociable under acid conditions, and reconstitution experiments provided evidence for the intermediate-sedimenting and viscous nature of the monoclonal IgA-polyclonal IgG complexes. This patient had no evident features of a lymphocytic or plasma cell malignancy or of a disease state typically associated with the presence of rheumatoid factor. Treatment with plasmaphereses and cyclophosphamide reduced the serum concentrations of the monoclonal IgA protein and the IgA-IgG immune complexes; this response was associated with decreased serum viscosity and attendant clinical improvement.     

Antibody‐dependent and antibody‐independent uptake of HBsAg across human leucocyte subsets is similar between individuals with chronic hepatitis B virus infection and healthy donors

Maintaining detectable levels of antibodies to hepatitis B surface antigen (HBsAg) in serum after HBsAg sero‐conversion is the key clinical endpoint indicative of recovery from infection with hepatitis B virus (HBV). As HBV‐infected hepatocytes secrete HBsAg subviral particles in vast excess over HBV virions, detectable hepatitis B surface antibody (anti‐HBs) titres imply complete elimination of HBV virions as well as HBsAg particles. Although intrahepatic phagocytes, for example Kupffer cells, are thought to mediate clearance of HBsAg via antibody (Ab)‐dependent and Ab‐independent mechanisms, the relative contributions of circulating phagocytic cell types to HBsAg elimination are poorly characterized. Understanding the role of various immune cell subsets in the clearance of HBsAg is important because Ab‐dependent or Ab‐independent phagocytic HBsAg uptake may modulate presentation of HBsAg‐derived epitopes to antigen‐specific T cells and hence plays a critical role in adaptive immunity against HBV. This study aims to characterize phagocytic leucocyte subsets capable of internalizing HBsAg immune complexes (HBsAg:IC) or un‐complexed HBsAg particles in whole blood directly ex vivo. The data show that uptake of HBsAg:IC occurs most prominently in monocytes, B cells, dendritic cells and in neutrophils. In contrast, B cells, and to a lesser degree also monocytes, seem to be effective phagocytes for un‐complexed HBsAg. Importantly, a similar pattern of phagocytic HBsAg uptake was observed in blood from chronic hepatitis B (CHB) patients compared to healthy controls, suggesting that phagocytosis‐related cellular functions are not altered in the context of CHB.     

In vitro synthesis of antibody to hepatitis B virus antigen by circulating lymphocytes from chronic HBsAg carriers and patients with acute hepatitis B

It is not clear what determines the outcome of hepatitis B surface antigen (HBsAg) infection, although evidence suggests that the absence of antibody to HBsAg (anti-HBs) is responsible for the development of the carrier state. The synthesis of immunoglobulin G (IgG), anti-HBs and antibody to hepatitis B core antigen (anti-HBc) was measured in pokeweed mitogen-stimulated cultures of peripheral blood mononuclear cells from 12 chronic HBsAg carriers, 5 patients with acute hepatitis B (AHB) during recovery phase and 11 subjects with anti-HBs in serum (controls). All 3 groups showed similar amounts of IgG synthesis. Anti-HBc was detectable in lymphocyte cultures of 10 of 12 chronic HBsAg carriers and 2 of 5 AHB patients, but in none of the controls. Anti-HBs was found in cultures from 6 of 11 controls, and not in carriers or AHB patients. Both in vitro anti-HBc and anti-HBs levels correlated significantly with serum titers of anti-HBc and anti-HBs respectively. B cells from controls cocultured with irradiated (helper) T cells from carriers and AHB patients produced anti-HBs normally. In contrast, B cells from 11 of 12 carriers and 3 of 5 AHB patients cocultured with irradiated control T cells did not synthesize detectable amounts of anti-HBs although they synthesized normal amounts of IgG and anti-HBc. T cells from 8 of 12 carriers and all AHB patients suppressed anti-HBs synthesis by mixtures of control B cells and control irradiated T cells, but these T cells did not affect IgG synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemagglutination and immunofluorescence studies on polymerized human serum albumin binding activity in chronic hepatitis B virus infection

The binding activity of polymerized human serum albumin was determined in 202 HBsAg carriers. The presence of polymerized human serum albumin receptor sites was tested by hemagglutination and differentiated from antihuman albumin antibodies by immunofluorescence, isolation of IgG and IgM fractions and testing of HBsAg anti-HBs immune complexes. A granular pattern with anti-HBs was specific for polymerized human serum albumin receptor sites as demonstrated with purified HBsAg. In addition, a linear pattern with fluoresceinated antihuman immunoglobulins might suggest the presence of antihuman albumin antibodies (which was generally due to an IgG antibody). However, a granular pattern with fluoresceinated antihuman immunoglobulins may indicate the presence of HBsAg anti-HBs immune complexes. A weak linear pattern was also observed simultaneously in these cases, probably due to IgM antihuman albumin antibodies or an antipolymerized human serum albumin receptor site antibody. Of 202 HBsAg-positive patients, 71 showed polymerized human serum albumin receptor sites activity. The highest percentage of polymerized human serum albumin receptor sites was found among patients showing HBeAg and hepatitis B virus DNA polymerase positivity (96%), followed by HBeAg positivity and hepatitis B virus DNA polymerase negativity (48%), and anti-HBe positivity and hepatitis B virus DNA polymerase negativity (17%). In addition, a significant correlation between polymerized human serum albumin titers and hepatitis B virus DNA polymerase was found (r = 0.573, p less than 0.01). However, at similar HBeAg titer, patients who were positive for hepatitis B virus DNA polymerase had a higher polymerized human serum albumin receptor sites titer than those who were negative for hepatitis B virus DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skin lesions in viral hepatitis: histologic and immunofluorescent findings.

A variety of skin rashes are knowned to occur as a part of the serum sickness-like prodrome of acute viral hepatitis which is thought to be due to immune complex deposition. We report the histologic and immunofluorescent findings in the skin and the seroloigc abnormalities in a patient with both erythematous maculopapular and purpuric rashes. We found circulating hepatitis B surface antigen (HBsAg), hypocomplementemia and cultaneous vasculitis associated with deposition of immunoglobulin and complement in the skin. We could not demonstrate intradermal deposition of HBsAg, but the findings are consistent with the immune complex hypothesis.     

Hepatitis B viral DNA in infected human liver and in hepatocellular carcinoma

Blot-hybridization analysis of DNA forms from hepatitis B virus (HBV) in the liver of chronic carriers of hepatitis B surface antigen (HBsAg) has revealed the presence of both relaxed and closed circular viral DNA and of novel viral DNA-RNA hybrid molecules in patients with complete virions, with the hepatitis B e antigen (HBeAg), or with both, in serum. One carrier of HBsAg with no detectable virus or HBeAg in his serum had small amounts of free viral DNA in his liver sample, a finding suggesting the potential for production of complete virus; another such carrier had only HBV DNA integrated in cellular DNA and, thus, may have lost the ability to replicate virus. The liver sample of one of eight patients with antibodies to HBsAg in his serum, but no HBsAg, contained small amounts of free viral DNA. Analysis of tissue from hepatocellular carcinoma revealed evidence for integrated viral DNA sequences in multiple-cellular DNA sites, and stoichiometric analysis suggested that the tumors were monoclonal in origin. These results demonstrate the presence of a new form of HBV in infected human liver and reveal that serological profiles are not always a reliable guide in determining the presence of potentially infectious forms of HBV in the liver.     

Lack of immune potentiation by complexing HBsAg in a heat-inactivated hepatitis B vaccine with antibody in hepatitis B immunoglobulin

In a randomized, dose-response study among 305 health care workers, we examined whether the immunogenicity of a heat-inactivated hepatitis B vaccine could be enhanced when HBsAg was complexed by anti-HBs contained in hepatitis B immunoglobulin either at equivalent proportions or at 10-fold antigen excess. The dose of HBsAg in the control vaccine as well as in the two complexed vaccine preparations could be reduced from the standard value (3 micrograms) to 0.6 micrograms per injection without affecting the antibody response in the vaccinees. Still lower dosages of HBsAg in the three vaccine preparations induced significantly lower but comparable anti-HBs responses. These results indicate that, in man, using a heat-inactivated plasma vaccine, addition of anti-HBs contained in hepatitis B immunoglobulin does not potentiate the immunogenicity of HBsAg.     

Selective functional deficit in dendritic cell--T cell interaction is a crucial mechanism in chronic hepatitis B virus infection

Summary. A defect in specific T cell immunity has long been assumed to be the central mechanism of persistent Hepatitis B virus (HBV) infection. Recent studies on HBV transgenic mice have suggested, however, that functional deficit of dendritic cells (DC) was an underlying cause for the T cell dysfunction. The functions of monocyte‐derived DC were determined by studying 75 subjects that included chronic hepatitis B patients with low or high HBV load; antibody to hepatitis B surface antigen (anti‐HBs) positive individuals who had recovered completely from previous acute HBV infection; healthy donors who had received hepatitis B vaccination and were anti‐HBs positive; and immunologically naïve to HBV or the vaccine individual. Impaired interactions between monocyte‐derived DC and T cells were shown in chronic HBV infection patients, especially in those with active virus replication. The dysfunctions included: (i) failure of DC to increase human leukocyte antigen (HLA‐II), B7 expression and interleukin‐12 secretion in responses to hepatitis B surface antigen (HBsAg), (ii) defective induction of T cell proliferative response to HBsAg, (iii) failure to activate T cells to produce cytokines and (iv) deficit in the induction of antigen specific cytotoxic T lymphocytes (CTLs). In vitro treatment of DC with tumour necrosis factor‐α improved HLA‐II and B7 expression, as well as Th cell and CTL responses. It is concluded that defective DC‐T cell interactions may account for the specific T cell immune defects in chronic HBV infection. Immunotherapy that aims at restoring DC functions could offer a new opportunity for effectively managing persistent HBV infections.     

Detection of HBeAg/anti-HBe immune complexes in the reactivation of hepatitis B virus replication among anti-HBe chronic carriers

Sixty-four chronic hepatitis B surface antigen (HBsAg) carriers with hepatitis B e antibody (anti-HBe) were followed in order to detect reactivations of hepatitis B virus (HBV) infection and to assess the incidence and specificity of hepatitis B e antigen/hepatitis B e antibody (HBeAg/anti-HBe) immune complexes (ICs). In 18 out of 19 patients who suffered an increase in alanine transaminase (ALT) values, serum HBV-DNA reappeared co-occurring with the peak(s) of transaminases. HBeAg/Anti-HBe immune complexes were detected in 17/18 (94.4%) patients positive for HBV-DNA. In nine of them, the appearance of immune complexes co-occurred with prednisone therapy, in two following seroconversion after recombinant interferon alpha-2A treatment, and spontaneously in the remaining seven patients. When ALT levels dropped to normal values, immune complexes as well as HBV-DNA became undetectable. In conclusion, the detection of HBeAg/anti-HBe immune complexes seems to be a specific method to detect HBV replication among anti-HBe positive patients.     

Preparation and characterization of human monoclonal antibodies directed against the hepatitis B virus surface antigen

The hepatitis B surface antigen (HBsAg) is highly immunogenic and induces an antibody response which is protective in vivo against hepatitis B virus (HBV) infection. Human monoclonal antibodies specific for HBsAg were produced, which could have potential therapeutic applications. Lymphocytes obtained from a vaccinated donor were stimulated in vitro and fused with the human myeloma cell line GM 4672, and eight hybridomas were obtained. Three of these clones, which reacted in an ELISA against the HBsAg vaccine, were expanded, subcloned and further analyzed. The subclones E7C2, C4C10, and D5B2 were able to bind to different HBsAg preparations, which express various subtypes, and recognized the major HBsAg peptides in Western blot analysis. Cross-inhibition experiments showed that E7C2, C4C10 and D5B2 are directed against the same epitope and have an affinity constant ranging from 5 X 10(7) to 3.3 X 10(9) M. Furthermore, these antibodies stained the surface and cytoplasm of the HBsAg-secreting cell lines PLC/PRF/5 and 4.10. The production of immunoglobulins varies from 0.3 to 1.3 micrograms/ml/10(6) and has remained stable over a period of 8 months. These human monoclonal antibodies, which appear to be directed against an antigenic determinant common to all HBsAg subtypes, could be useful in the study of HBV-related liver diseases as well as in their diagnosis and experimental therapy.     

Relationship of pre-S encoded antigens in liver and clinical manifestations of chronic hepatitis B infection

Pre-S1 and pre-S2 encoded antigens of hepatitis B virus were localized in liver tissue using monoclonal antibodies. They were found to be exclusively expressed in the cytoplasm of liver cells. Cell bound pre-S1 encoded protein was often detected in patients with chronic liver disease and viremia. Only a small number of the HBsAg positive cells also contained pre-S1 antigen. There was no correlation with nuclear HBcAg. Livers of non-viremic HBsAg carriers contained many HBsAg expressing liver cells, that were frequently also positive for pre-S2 encoded protein but contained no detectable pre-S1 encoded protein at all. It remains open whether cell bound pre-S2 containing proteins of middle size have a significance for pathogenesis, as they are present in individuals with chronic liver disease as well as in healthy HBsAG carriers, and may be associated with both increased and normal liver enzymes. Cell bound pre-S1 antigen with viremia may, however, be involved in the maintenance of viremia and liver disease.