An unexpected intriguing effect of Toll-like receptor regulator RP105 (CD180) on atherosclerosis formation with alterations on B-cell activation.

OBJECTIVE: In atherosclerosis, Toll-like receptors (TLRs) are traditionally linked to effects on tissue macrophages or foam cells. RP105, a structural TLR4 homolog, is an important regulator of TLR signaling. The effects of RP105 on TLR signaling vary for different leukocyte subsets known to be involved in atherosclerosis, making it unique in its role of either suppressing (in myeloid cells) or enhancing (in B cells) TLR-regulated inflammation in different cell types. We aimed to identify a role of TLR accessory molecule RP105 on circulating cells in atherosclerotic plaque formation. APPROACH AND RESULTS: Irradiated low density lipoprotein receptor deficient mice received RP105(-/-) or wild-type bone marrow. RP105(-/-) chimeras displayed a 57% reduced plaque burden. Interestingly, total and activated B-cell numbers were significantly reduced in RP105(-/-) chimeras. Activation of B1 B cells was unaltered, suggesting that RP105 deficiency only affected inflammatory B2 B cells. IgM levels were unaltered, but anti-oxidized low-density lipoprotein and anti-malondialdehyde-modified low-density lipoprotein IgG2c antibody levels were significantly lower in RP105(-/-) chimeras, confirming effects on B2 B cells rather than B1 B cells. Moreover, B-cell activating factor expression was reduced in spleens of RP105(-/-) chimeras. CONCLUSIONS: RP105 deficiency on circulating cells results in an intriguing unexpected TLR-associated mechanisms that decrease atherosclerotic lesion formation with alterations on proinflammatory B2 B cells.

T-cell co-stimulation by CD28-CD80/86 and its negative regulator CTLA-4 strongly influence accelerated atherosclerosis development.

OBJECTIVE: T-cells are central to the immune response responsible for native atherosclerosis. The objective of this study is to investigate T-cell contribution to post-interventional accelerated atherosclerosis development, as well as the role of the CD28-CD80/86 co-stimulatory and Cytotoxic T-Lymphocyte Antigen (CTLA)-4 co-inhibitory pathways controlling T-cell activation status in this process. METHODS AND RESULTS: The role of T-cells and the CD28-CD80/86 co-stimulatory and CTLA-4 co-inhibitory pathways were investigated in a femoral artery cuff mouse model for post-interventional remodeling, with notable intravascular CTLA-4+ T-cell infiltration. Reduced intimal lesions developed in CD4(-/-) and CD80(-/-)CD86(-/-) mice compared to normal C57Bl/6J controls. Systemic abatacept-treatment, a soluble CTLA-4Ig fusion protein that prevents CD28-CD80/86 co-stimulatory T-cell activation, prevented intimal thickening by 58.5% (p=0.029). Next, hypercholesterolemic ApoE3*Leiden mice received abatacept-treatment which reduced accelerated atherosclerosis development by 78.1% (p=0.040) and prevented CD4 T-cell activation, indicated by reduced splenic fractions of activated KLRG1+, PD1+, CD69+ and CTLA-4+ T-cells. This correlated with reduced plasma interferon-γ and elevated interleukin-10 levels. The role of CTLA-4 was confirmed using CTLA-4 blocking antibodies, which strongly increased vascular lesion size by 66.7% (p=0.008), compared to isotype-treated controls. CONCLUSIONS: T-cell CD28-CD80/86 co-stimulation is vital for post-interventional accelerated atherosclerosis development and is regulated by CTLA-4 co-inhibition, indicating promising clinical potential for prevention of post-interventional remodeling by abatacept.

Hyperfibrinolytic activity in hospitalized cirrhotic patients in a referral liver unit.

OBJECTIVE: Increased frequency of hyperfibrinolytic activity was reported in patients with cirrhosis. However, the incidence, clinical presentation, and the parameters related to hyperfibrinolysis remain largely unknown in these patients. By utilizing euglobulin lysis time (ELT) and other clinical coagulation tests, the present study investigated the incidence of and clinical parameters related to hyperfibrinolytic activity, and assessed predicting factors to epsilon-aminocaproic acid (EACA) treatment in cirrhotic patients with hyperfibrinolysis in a liver unit. METHODS: The study included 86 consecutive patients who were referred and admitted to a referral liver unit for various liver diseases. The mean age was 50.0 yr, with a male: female ratio of 60:26. Sixty-six patients (76.7%) were Hispanic and 75 (87.2%) were cirrhotic. The etiologies of liver diseases included alcoholic liver disease (n = 68, 79.1%), hepatitis B (n = 2, 2.3%), hepatitis C (n = 6, 7.0%), autoimmune hepatitis (n = 3, 3.5%), cryptogenic liver disease (n = 4, 4.7%), and hepatocellular carcinoma (n = 3, 3.5%). Coagulation studies included ELT, PT, PTT, fibrinogen, D-dimer, and fibrin degradation product levels. RESULTS: Hyperfibrinolytic activity as reflected by shortened ELT was present in 27/75 cirrhotic (31.3%) but 0/11 noncirrhotic patients, which was significantly correlated with higher Child-Pugh (C-P) class, abnormal levels of PT, PTT, fibrinogen, platelet count, and total bilirubin. Shortened ELT was more frequently seen in patients with hepatic decompensation and mucocutaneous bleeding, although these relationships were not statistically significant. In 27 patients with hyperfibrinolysis, five (18.5%) required EACA treatment for progressive mucocutaneous bleeding and/or hematoma. EACA treatment was significantly associated with higher C-P scores; greatly shortened ELT (< or =50% of normal value); and abnormal levels of fibrinogen, total bilirubin, and PT, indicating that these factors may serve as predictors for EACA treatment. CONCLUSION: Hyperfibrinolytic activity was seen in 31.3% of patients with cirrhosis, which is correlated with higher C-P scores; abnormal PT, PTT, fibrinogen level, and platelet count; and hyperbilirubinemia. Patients who received EACA treatment usually have a more severe hyperfibrinolytic activity as indicated by shortened ELT and low level of fibrinogen, and more severe liver disease as indicated by higher C-P scores and hyperbilirubinemia.

Long lasting p53-specific T cell memory responses in the absence of anti-p53 antibodies in patients with resected primary colorectal cancer.

Colorectal carcinoma is commonly associated with mutation and overexpression of p53, making this antigen a potential target for immune intervention. We analyzed humoral and proliferative immunity against p53 in the blood of patients with resected primary colorectal cancer. The majority of these patients displayed anti-p53 T helper (Th) immunity in the absence of measurable p53 specific antibody levels. The Th responses were long-lasting since they could be detected up to several years after resection of the primary tumor. In a number of cases the Th responses were highly sensitive, reflected by the recognition of naturally processed p53 protein. Our data argue that boosting of these responses in patients with minimal residual disease through p53-specific vaccination, may be employed for improving the chance of disease-free survival of these patients.

Acute hepatitis induced by bupropion.

As an antidepressant, bupropion is considered to be a safe agent that usually causes infrequent and mild increase of serum liver enzymes. Asymptomatic elevation of serum transaminases was previously reported only in a single case. We describe a patient who developed typical acute hepatitis after receiving six weeks of bupropion for depression. His presentation was characterized with acute onset of symptoms associated with significantly elevated ALT, AST, and LDH and acute hepatic inflammation. The clinical course of our patient, including incubation period, pattern of liver enzyme elevation, and time of recovery, was similar to, but much more severe than, the case reported by Oslin and Duffy. Discontinuation of bupropion was followed by a rapid resolution of clinical symptoms and liver enzymes. The incidence of bupropion-induced hepatitis remains to be defined even though it appears to be relatively low. Since the clinical application of bupropion is broader, we must be aware of the clinical entity of bupropion-induced hepatitis.

A pilot randomized, controlled trial of the effect of iron depletion on long-term response to alpha-interferon in patients with chronic hepatitis C.

BACKGROUND/AIMS: Some studies have suggested that hepatic iron may influence the response to interferon therapy in chronic hepatitis C patients. We conducted this randomized, controlled trial to evaluate the effect of iron depletion on: (1) aminotransferase activity and hepatitis C RNA levels; and (2) response to interferon therapy in 38 patients with elevated alanine aminotransferase levels and who were HCV RNA positive. METHODS: Seventeen patients underwent a 500-ml phlebotomy every 2 weeks until iron deficiency was achieved. Patients were then started on a 6-month course of alpha-interferon 2b (3 mu tiw). Controls were 21 patients who were monitored for a 6- to 8-week period without phlebotomy prior to interferon therapy. Response to interferon was defined as loss of serum HCV RNA by reverse transcriptase-polymerase chain reaction. Serum HCV RNA was quantitated by bDNA technique. RESULTS: Alanine aminotransferase levels decreased in 15/17 patients after phlebotomy. Mean alanine aminotransferase fell from 156.8 to 89.7 U/l (p=0.008). Changes in iron indices and alanine aminotransferase after phlebotomy were not accompanied by changes in HCV RNA levels. In control patients, neither alanine aminotransferase nor HCV RNA levels changed during the observation period. At the end of 24 weeks of interferon therapy, 7/17 phlebotomized patients had a response, compared to 6/21 control patients (p=ns). After 6 months of follow-up, 5/17 phlebotomized patients remained HCV RNA negative, in contrast to only 1/21 controls (p=0.07). CONCLUSIONS: Iron depletion led to a reduction in aminotransferase levels; this was not accompanied by changes in levels of hepatitis C RNA. There may be an improvement in the sustained response to interferon therapy, but this requires confirmation.

Effect of iron depletion on long-term response to interferon-alpha in patients with chronic hepatitis C who previously did not respond to interferon therapy.

About half of patients with chronic hepatitis C treated with interferon will not have a biochemical or virological response. Several studies suggested that increased hepatic iron content may negatively influence the response to interferon. We conducted this prospective trial to evaluate the effect of iron depletion on the response to a repeat course of interferon in 20 chronic hepatitis C patients who previously had not responded to interferon. The patients underwent 500-ml phlebotomies every 2 weeks until iron deficiency was achieved. Patients were then started on a 6-month course of interferon alfa-2b (3 million units, t.i.w.). These patients required a mean of 6.0 (range, 1-14) phlebotomies to become iron deficient. ALT levels decreased in 18 of 20 patients and became normal in 4 patients. Mean ALT levels decreased from 154.2 to 87.9 U/L (p = 0.0006). At the end of 24 wk of interferon therapy, ALT levels were normal in 11 patients, 3 of whom had undetectable HCV RNA in the serum. One additional patient with abnormal ALT had undetectable HCV RNA. After 6 months of follow-up, one of the HCV RNA negative patients relapsed with reappearance of HCV RNA and elevation of ALT. In summary, 15% of chronic hepatitis C patients who previously failed interferon now had a sustained response to interferon therapy that was preceded by iron depletion.

Clinical significance of hepatitis C viral RNA status and its correlation to antibodies to structural HCV antigens in anti-HCV reactive patients with normal liver tests.

Extensive serological testing and HCV RNA determination by RT-PCR was performed in serum, PBMCs, and liver tissue in thirteen anti-HCV reactive patients with persistently normal liver tests. Absolute concordance in the status of HCV RNA between serum, PBMCs, and liver was noted. Five patients were HCV RNA positive but only three had mild histological changes. Eight patients were HCV RNA negative in all three sites and had virtually normal liver histology. Patterns of reactivity in RIBA 2.0 strip immunoblot assay did not differentiate viremic from nonviremic patients. ELISA testing using multiple individual HCV recombinant antigens from the structural and non-structural regions of HCV demonstrated mean antibody titers to the structural antigens, in particular HCV E2 antibodies, to be significantly lower in HCV RNA negative patients. The status of HCV RNA in the serum appears to infer the status of HCV RNA in the liver and PBMCs in patients with persistently normal liver tests. Patients with persistently normal liver tests and undetectable HCV RNA have probably spontaneously cleared HCV infection.

HLA A2 restricted cytotoxic T lymphocyte responses to multiple hepatitis B surface antigen epitopes during hepatitis B virus infection.

Inasmuch as the hepatitis B virus (HBV) is not directly cytopathic for the infected hepatocyte, it is generally presumed that viral clearance and liver cell injury during viral hepatitis are due to a CTL response to HBV encoded Ag presented by HLA class I molecules. We have previously examined the peripheral blood CTL response to two HBV nucleocapsid epitopes in patients with acute and chronic viral hepatitis, one of which is restricted by HLA-A2, whereas the other is dually restricted by HLA-A31 and Aw68. In this study, we defined the HLA-A2-restricted CTL response to the hepatitis B surface Ag (HBsAg) by using a panel of HBsAg-derived synthetic peptides containing the ideal HLA-A2.1 binding motif (-L------V). Several novel aspects of HBV immunobiology and pathogenesis are evident from this study. First, the peripheral blood CTL response to HBV-encoded Ag is remarkably polyclonal and multispecific in most patients with acute hepatitis. Indeed, HLA-A2-restricted CTL specific for as many as four envelope epitopes and one nucleocapsid epitope were found to be present simultaneously in individual patients with acute viral hepatitis. Second, HBV-specific CTL are not detectable in the peripheral blood in a minority of patients with acute hepatitis, nor have we detected a CTL response in any of the patients with chronic hepatitis that we have studied thus far. Although the cellular and molecular basis for CTL nonresponse remains to be determined, the data suggest that it may contribute to viral persistence. Third, the diversity and the specificity of the CTL response is determined in part by the coding sequence of the viral genome present in each infected patient. Indeed, the apparent nonresponse of some acutely infected patients to at least one HBsAg-specific CTL epitope actually reflects infection by a viral variant that contains a critical substitution in one of the anchor residues within the epitope. Finally, at a fundamental level, the data suggest that the presence of the HLA-A2.1-binding motif in a peptide may not be sufficient for binding; and the capacity of a peptide to bind the class I molecule does not guarantee that it will be immunogenic.

HLA-A31- and HLA-Aw68-restricted cytotoxic T cell responses to a single hepatitis B virus nucleocapsid epitope during acute viral hepatitis.

We have recently developed the technology to identify and characterize the human histocompatibility leukocyte antigen (HLA) class I-restricted, CD8+ cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV)-encoded antigens in patients with acute viral hepatitis. CTL are expanded in vitro by stimulation with HBV-derived synthetic peptides and selected by restimulation with a panel of HLA-matched stable transfectants that express the corresponding HBV protein. We have recently reported the existence of an HLA-A2-restricted, CD8+ CTL response to an epitope located between residues 18 and 27 of the HBV nucleocapsid core antigen (HBcAg). We now report the discovery of a CTL epitope located between HBcAg residues 141 and 151 that completely overlaps a critical domain in the viral nucleocapsid protein that is essential for its nuclear localization and genome packaging functions as well as processing of the precore protein. The CTL response to this epitope is dually restricted by the HLA-A31 and HLA-Aw68 alleles, which, unexpectedly, appear to use a common binding motif based on the results of alanine substitution and competition analysis, and the binding properties of these two alleles predicted from their known primary sequence, and from the three-dimensional structure of HLA-Aw68. We have also demonstrated that the HBV-specific CTL response to this epitope is polyclonal during acute viral hepatitis, since these two restriction elements can present the HBcAg 141-151 epitope to independent CTL clones derived from a single patient; and that the CTL response is multispecific, since HLA-A2-restricted and HLA-Aw68-restricted CTL responses to HBcAg 18-27 and HBcAg 141-151, respectively, have been identified to coexist in another patient. The foregoing argue against the emergence of CTL escape mutants as a significant problem during HBV infection, especially at this locus, where mutations might be incompatible with viral replication. Finally, our data suggest an association between the HBV-specific CTL response and viral clearance, and they have implications for the design of immunotherapeutic strategies to terminate HBV infection in chronically infected patients.

Hepatocellular carcinoma in a case of Wilson's disease.

Hepatocellular carcinoma has rarely been reported in Wilson's disease, particularly in women. We describe the case of a female patient who was diagnosed with Wilson's disease at the age of 39 years, after presenting with severe neurological symptoms. She had significant neurological improvement following penicillamine therapy and succumbed to hepatocellular carcinoma at the age of 72 years, following 33 years of penicillamine therapy. The patient described here was the oldest and only the third female patient with hepatocellular carcinoma complicating Wilson's disease to be reported in the literature.

Interactions of HDV, HBV and HIV in chronic B and D infections and in reactivation of chronic D infection.

From this study, we can conclude that there is significant influence of HIV infection on the clinical course of chronic HDV as follows: In these patients, there is simultaneous replication of both HBV and HDV and the suppression of HBV by HDV is modified. There is decreased antibody response to HDV, however, the degree of liver injury is not altered. Although these patients tend to have "reactivation episodes" as frequently as the HIV negative group, no correlation of serum ALT to HDV-RNA could be found. The possibility of these episodes resulting from injury due to other viruses such as non-A, non-B cannot be excluded.

Loss of heterozygosity suggests tumor suppressor gene responsible for primary hepatocellular carcinoma.

Primary hepatocellular carcinoma (PHC), epidemiologically associated with chronic hepatitis B virus (HBV) infection, has historically been felt to be caused by the activation or introduction of an oncogene. However, transforming sequences from human PHC have not been reproducibly isolated. In this paper, evidence is presented that suggests PHC may result instead from the loss of an anti-oncogene. Seven of 12 human primary liver tumors tested against a panel of restriction fragment length polymorphisms (RFLPs) demonstrated loss of constitutional heterozygosity for markers on chromosome 4. Tumor and nontumor liver tissue were typed for 11 chromosome 4 RFLPs. In addition, at least one RFLP on nine other chromosomes (1, 2, 6, 7, 9, 11, 13, 14, and 17) was tested for allelic loss. Seven of nine tumors constitutionally heterozygous for chromosome 4q markers showed allele loss in tumor tissue. Six of the seven samples were jointly informative for both 4p and 4q markers. Five of the six demonstrated loss for only 4q RFLPs. In one individual, in which two samples were taken from distant locations within the same tumor, both samples showed loss of the same alleles. Among the other chromosomes informative for allele loss, one tumor showed changes on 13q. No other changes were observed in RFLPs located on the eight other chromosomes tested. These results indicate that an anti-oncogene may be located on 4q and suggest a mechanism for PHC and other cancers seroepidemiologically related to virus infection. Liver cancer caused by chronic HBV infection or other environmental agents may be linked through genetic events responsible for the loss of a tumor suppressor locus (anti-oncogene) located on chromosome 4.

Spontaneous rupture of nonparasitic cyst of the liver.

A case of spontaneous rupture of a nonparasitic liver cyst, documented by serial computed tomography scans, is reported. The patient was closely followed clinically after the rupture of the cyst and was treated conservatively, without surgical intervention. No peritonitis developed. This unusual complication of nonparasitic simple liver cysts may not always require surgery.

Correlation of IgM anti-hepatitis D virus (HDV) to HDV RNA in sera of chronic HDV.

One hundred forty-four serum samples from 52 patients with chronic hepatitis D virus infection were analyzed for hepatitis D virus RNA by dot-blot hybridization using hepatitis D virus cDNA probe labeled with 32P. The results were correlated with the presence of serum IgM anti-hepatitis D virus and hepatitis D antigen in liver biopsy specimens when available. Although there was a trend of positive correlation between serum hepatitis D virus RNA and IgM anti-hepatitis D virus, no statistical significance could be found. In the serum samples with hepatitis D virus RNA, 32% were found to be negative for IgM anti-hepatitis D virus. Therefore, in chronic hepatitis D virus, absence of IgM anti-hepatitis D virus does not rule out active viral infection, as suggested by previous studies. There was a strong correlation between serum hepatitis D virus RNA and hepatic hepatitis D virus antigen. These data indicate that detection of hepatitis D virus RNA in serum samples is a reliable noninvasive marker of active viral infection.

An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis.

A specific assay has been developed for a blood-borne non-A, non-B hepatitis (NANBH) virus in which a polypeptide synthesized in recombinant yeast clones of the hepatitis C virus (HCV) is used to capture circulating viral antibodies. HCV antibodies were detected in six of seven human sera that were shown previously to transmit NANBH to chimpanzees. Assays of ten blood transfusions in the United States that resulted in chronic NANBH revealed that there was at least one positive blood donor in nine of these cases and that all ten recipients seroconverted during their illnesses. About 80 percent of chronic, post-transfusion NANBH (PT-NANBH) patients from Italy and Japan had circulating HCV antibody; a much lower frequency (15 percent) was observed in acute, resolving infections. In addition, 58 percent of NANBH patients from the United States with no identifiable source of parenteral exposure to the virus were also positive for HCV antibody. These data indicate that HCV is a major cause of NANBH throughout the world.

Status of hepatitis B virus DNA in alcoholic liver disease: a study of a large urban population in the United States.

Two reports have shown hepatitis B virus DNA in serum and liver tissue in alcoholic liver disease with negative serum HBsAg, suggesting a pathogenetic role for hepatitis B virus. We studied hepatitis B virus DNA in serum and liver from three groups of alcoholic patients; (Group 1) 50 patients without liver disease, (Group 2) 108 patients with alcoholic liver disease and (Group 3) five patients with alcoholic liver disease and hepatocellular carcinoma. Serum was tested for HBsAg, anti-hepatitis B core and anti-hepatitis B surface by radioimmunoassay and hepatitis B virus DNA by direct spot hybridization. Liver tissue from Groups 2 and 3 (113 patients) was examined by Southern blot analysis using 32P-labeled hepatitis B virus DNA clone from pBR322. Controls were 21 patients with chronic hepatitis B virus (14 patients with chronic active hepatitis, seven patients with cirrhosis and hepatocellular carcinoma). Serum and tissue were analyzed for hepatitis B virus DNA. Hepatitis B virus DNA was not detected in either serum or liver tissue in any of the 163 patients (Groups 1 to 3). In contrast, among the controls, hepatitis B virus DNA was present in the serum of 15 of the 21. Tissue DNA in those with chronic active hepatitis revealed 10/14 with free hepatitis B virus DNA, two with integrated sequences and two with no viral sequences. All seven patients with hepatocellular carcinoma had integrated viral DNA sequences in the tumor tissues. From these results, it appears that hepatitis B virus does not play a role in the pathogenesis of alcoholic liver disease.

Permanent HBsAg clearance in chronic hepatitis B viral infection following acute delta superinfection.

Three patients are described with chronic hepatitis B virus infection for three to six years before hepatitis delta virus superinfection occurred. Liver biopsy performed in two patients prior to their delta illness revealed chronic persistent hepatitis and chronic active hepatitis, respectively. Within one to seven months of the acute delta event, all three patients lost their circulating hepatitis B surface antigen. Subsequently, delta antibody also cleared. Clinical well-being and normal transaminases were documented over 10-44 months of follow-up. Although most cases of delta infection in chronic hepatitis B result in severe or progressive disease, a small number of patients may develop clearance of the HBsAg with clearance of both B and delta infections.

Markers of viral replication in patients with chronic hepatitis B virus infection.

Serum samples from 56 patients with biopsy-proven chronic B viral hepatitis without superimposed delta hepatitis were analyzed for the various markers of viral replication, including serum hepatitis B e Ag (HBeAg), hepatitis B virus deoxyribonucleic acid (HBV-DNA), and hepatitis B core antigen (HBcAg) in the liver tissues. Twenty-seven patients had persistent viral hepatitis (PH) and 29 patients had chronic active hepatitis (CAH) with or without cirrhosis. HBV-DNA was identified in the sera of 81% of patients with PH and 60% of patients with CAH. Significantly higher levels of HBV-DNA were found in patients with PH than in those with CAH. Both HBeAg in serum and HBcAg in liver correlated positively with serum HBV-DNA. Nine patients had serum HBV-DNA in the absence of HBeAg (four had anti-HBe), and seven of these nine patients had stainable HBcAg in the liver (two did not have staining). None of these patients had hepatic HBcAg in the absence of serum HBV-DNA. When these patients were stratified according to their epidemiologic background, serum HBV-DNA was present in a significantly higher number of male homosexuals than in any other groups. This was unrelated to their status of human immunodeficiency viral serology.

A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 delta and p27 delta.

On the basis of the complete nucleotide sequence of the single-stranded, covalently closed circular hepatitis delta virus RNA genome (K.-S. Wang, Q.-L. Choo, A. J. Weiner, J.-H. Ou, R. C. Najarian, R. M. Thayer, G. T. Mullenbach, K. J. Denniston, J. L. Gerin, and M. Houghton, Nature [London] 323:508-514, 1986 [Author's correction, 328:456, 1987]), five long open reading frames (ORFs) encoding polypeptides containing a methionine proximal to the amino terminus were expressed in bacteria. Only polypeptides encoded by the antigenomic ORF5 cross-reacted with antisera obtained from patients with hepatitis delta virus infections. Immunological analysis of viral extracts and the recombinant ORF5 polypeptides synthesized in bacteria and yeast cells revealed that ORF5 encodes the immunogenic epitope(s) shared by both hepatitis delta viral polypeptides p27 delta and p24 delta and probably represents the complete structural gene for p27 delta and p24 delta. We also present evidence that ORF5 encodes the hepatitis delta antigen, an antigen originally found in the nuclei of hepatocytes of infected individuals (M. Rizzetto, M. G. Canese, S. Arico, O. Crivelli, F. Bonino, C. G. Trepo, and G. Verme, Gut 18:997-1003, 1977). A comparison of the primary structure of the predicted hepatitis delta antigen polypeptides with that of the core antigen of the hepatitis B virus shows that these polypeptides are very dissimilar.