Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies.

Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV-genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1-48 of the pre-S1 domain, fused to S (pre-S1.1-48/S). The antigen loop in S protein and the selected pre-S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 10(7) rSFV particles and 10(8) rSFV particles 3 weeks later. Antibodies induced by rSFV encoding S proteins reacted preferentially with subtype determinants of yeast-derived S antigen but equally well with patient-derived S antigen. Immunization with rSFV encoding pre-S1.1-48/S resulted in formation of pre-S1- and S-specific immunoglobulin G (IgG), while immunization with the isogenic mutant without S start codon induced pre-S1 antibodies only. Neutralizing antibodies were determined by mixing with plasma-derived HBV/ayw2 and subsequent inoculation of susceptible primary hepatocyte cultures from Tupaia belangeri. S/adw2 antisera neutralized HBV/ayw2 as effectively as antisera raised with S/ayw2. The pre-S1 antibodies also completely neutralized HBV infectivity. The IgG1/IgG2a ratios ranged from 0.28 to 0.88 in the four immunized groups and were lowest for the pre-S1.1-48/S vector, indicating the strongest Th1 response. This vector type may induce subtype-independent and S-escape-resistant neutralizing antibodies against HBV.

Tissue donation and virus safety: more nucleic acid amplification testing is needed.

In tissue and organ transplantation, it is of great importance to avoid the transmission of blood-borne viruses to the recipient. While serologic testing for anti-human immunodeficiency virus (HIV)-1 and -2, anti-hepatitis C virus (HCV), hepatitis B surface antigen (HBsAg), anti-hepatitis B core antigen (HBc), and Treponema pallidum infection is mandatory, there is until now in most countries no explicit demand for nucleic acid amplification testing (NAT) to detect HIV, hepatitis B virus (HBV), and HCV infection. After a review of reports in the literature on viral transmission events, tissue-specific issues, and manufacturing and inactivation procedures, we evaluated the significance of HIV, HCV, and HBV detection using NAT in donors of various types of tissues and compared our results with the experiences of blood banking organizations. There is a significant risk of HIV, HCV, and HBV transmission by musculoskeletal tissues because of their high blood content and the high donor-recipient ratio. If no effective virus inactivation procedure for musculoskeletal tissue is applied, donors should be screened using NAT for HIV, HCV, and HBV. Serologically screened cardiovascular tissue carries a very low risk of HIV, HCV, or HBV transmission. Nevertheless, because effective virus inactivation is impossible (retention of tissue morphology) and the donor-recipient ratio may be as high as 1:10, we concluded that NAT should be performed for HIV, HCV, and HBV as an additional safety measure. Although cornea allografts carry the lowest risk of transmitting HIV, HCV, and HBV owing to corneal physiology, morphology, and the epidemiology of corneal diseases, NAT for HCV should still be performed. If the NAT screening of a donor for HIV, HCV, and HBV is negative, quarantine storage of the donor tissue seems dispensable. In view of numerous synergistic effects with transfusion medicine, it would be advantageous for tissue banks to cooperate with blood bank laboratories in performing virological tests.

Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex.

Although many viruses replicate in the nucleus, little is known about the processes involved in the nuclear import of viral genomes. We show here that in vitro generated core particles of human hepatitis B virus bind to nuclear pore complexes (NPCs) in digitonin-permeabilized mammalian cells. This only occurred if the cores contained phosphorylated core proteins. Binding was inhibited by wheat germ agglutinin, by antinuclear pore complex antibodies, and by peptides corresponding either to classical nuclear localization signals (NLS) or to COOH-terminal sequences of the core protein. Binding was dependent on the nuclear transport factors importins (karyopherins) alpha and beta. The results suggested that phosphorylation induces exposure of NLS in the COOH-terminal portion of the core protein that allows core binding to the NPCs by the importin- (karyopherin-) mediated pathway. Thus, phosphorylation of the core protein emerged as an important step in the viral replication cycle necessary for transport of the viral genome to the nucleus.

Core promoter mutant HBV non-responding to adefovir after viral breakthrough on lamivudine: rapid virologic response to tenofovir plus lamivudine in a cirrhotic patient.

BACKGROUND: In chronic hepatitis B patients undergoing therapy with LAM or ADV, viral breakthrough is possible due to the emergence of drug resistance. LAM resistant HBV strains are susceptible to ADV, while ADV resistant mutants remain sensitive to LAM. CASE REPORT: A male patient with HBV-related cirrhosis developed viral breakthrough (HBV DNA>1.8 x 106 IU/ml) after 4 1/2 years of treatment with LAM, and therapy was switched to ADV (10 mg/d). After three months, HBV remained highly replicative without any changes of ALT values, and ADV dose was increased (20 mg/d). Because of unchanged VL sequence analysis was performed three months later, which showed the mutation (rtS219A) and the concomitant mutation (sS210R) and 2 mutations in core promoter region (A1762T), (G1764A). During the sixth month of ADV monotherapy the patient developed liver failure. After administration of TDF plus LAM, HBV DNA became undetectable within 39 days. At day 41, the patient underwent OLT. TDF plus LAM were well tolerated, and the patient maintained undetectable HBV DNA levels, and in addition to HBIG a sustained HBsAg negative status over twenty-eight months post OLT. CONCLUSION: TDF plus LAM is a safe drug combination in case of viral breakthrough during LAM treatment and subsequent primary non-response to ADV. High VL persisting for >or= 6 months of continuous antiviral treatment may indicate drug resistance. Especially in cirrhotic patients with LAM resistance, "add on" of a nucleotide analogue is the right therapeutic strategy even before viral breakthrough gets apparent.

Induction of apoptosis by the transactivating domains of the hepatitis B virus X gene leads to suppression of oncogenic transformation of primary rat embryo fibroblasts.

Epidemiology shows a clear correlation between chronic infection with the hepatitis B virus (HBV) and development of hepatocellular carcinoma (HCC). The potential role of the transactivating hepatitis B virus X protein (HBx) in transformation by HBV is controversial. Here we report that HBx suppresses transformation of primary rat embryo fibroblasts (REFs). Cooperating oncogenes like c-Ha-ras and c-myc transform REF very efficiently but cotransfection with HBx suppressed transformation of REFs down to 5%. Similarly, transfection of HBx together with the cooperating oncogenes Ha-ras and SV40 LTAg or c-Ha-ras and mutant p53 reduced the number of foci to 13%. Comparable results were obtained with HBx in the context of the whole HBV. Suppression of focus formation in REF could be partly relieved by cotransfection of apoptosis inhibitors Bcl-2 or E1B. However, cotransfection of apoptosis inhibitors crmA and p35 did not influence the proapoptotic functions of HBx. Thus, HBx may specifically activate the Bcl-2 sensitive pathway leading to apoptosis. Experiments with 13 HBx linker scanning mutants revealed that the domains necessary for HBx dependent transactivation overlap with the domains needed for the apoptotic/growth arrest functions of HBx.

Hemifusion activity of a chimeric influenza virus hemagglutinin with a putative fusion peptide from hepatitis B virus.

Entry of enveloped viruses is often mediated by an aminoterminal hydrophobic fusion peptide of a viral surface protein. The S domain of the hepatitis B virus surface protein contains a putative fusion peptide at position 7-18, but no systems are available to study its function directly. We tested the functionality of this peptide and a related peptide from another hepadnavirus in the context of the well-characterized influenza virus hemagglutinin H7 using gene mutation. The chimeric hemagglutinins could be expressed stably in CV 1 cells and were transported to the cell surface. The chimeras were incompletely cleaved by cellular proteases but cleavage could be completed by trypsin treatment of the cells. The chimeras did not differ in receptor binding, i.e. erythrocyte binding. Hemifusion and fusion pore formation were detected with membrane or cytosolic fluorescent dye-labeled erythrocytes as target structures of the hemagglutinin. Five of six different chimeras mediated hemifusion in 20-54% of the hemagglutinin-expressing cells, complete fusion and syncytium formation was not observed. The data suggest that the sequence 7-18 of the hepatitis B S domain may indeed initiate the first step of viral entry, i.e. hemifusion.

Morphological and functional effects of antisense RNA to the deleted in colorectal carcinoma (DCC) gene in a pancreatic carcinoma cell line.

To investigate the role of the deleted in colorectal carcinoma gene (DCC) in cells of pancreatic origin (MiaPaCa-2) we established cell lines stably expressing DCC antisense RNA. Expression of DCC antisense RNA led to striking alterations in the MiaPaCa-2 cell line. Antisense transfectants had nearly lost adherence and had acquired a spherical morphology. The ordered structure of actin bundles in the parental cell line had been lost largely in DCC antisense RNA expressing cell clones. Moreover, the antisense DCC transfected cells displayed a decreased growth rate, a decrease of cells in G1 phase and an accumulation in S phase of the cell cycle. These heavily altered characteristics of MiaPaCa-2 cells expressing DCC antisense RNA point to a yet unknown role for DCC in an important intracellular pathway.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in health care workers (HCWs): guidelines for prevention of transmission of HBV and HCV from HCW to patients.

The transmission of viral hepatitis from health care workers (HCW) to patients is of worldwide concern. Since the introduction of serologic testing in the 1970s there have been over 45 reports of hepatitis B virus (HBV) transmission from HCW to patients, which have resulted in more than 400 infected patients. In addition there are six published reports of transmissions of hepatitis C virus (HCV) from HCW to patients resulting in the infection of 14 patients. Additional HCV cases are known of in the US and UK, but unpublished. At present the guidelines for preventing HCW to patient transmission of viral hepatitis vary greatly between countries. It was our aim to reach a Europe-wide consensus on this issue. In order to do this, experts in blood-borne infection, from 16 countries, were questioned on their national protocols. The replies given by participating countries formed the basis of a discussion document. This paper was then discussed at a meeting with each of the participating countries in order to reach a Europe-wide consensus on the identification of infected HCWs, protection of susceptible HCWs, management and treatment options for the infected HCW. The results of that process are discussed and recommendations formed. The guidelines produced aim to reduce the risk of transmission from infected HCWs to patients. The document is designed to complement existing guidelines or form the basis for the development of new guidelines. This guidance is applicable to all HCWs who perform EPP, whether newly appointed or already in post.

HBc and HBe specificity of monoclonal antibodies against complete and truncated HBc proteins from E. coli.

We have prepared and used monoclonal antibodies against various populations of full-length and truncated hepatitis B core proteins in order to distinguish between epitopes of HBcAg and HBeAg. Our results show that various epitopes are specific for the different proteins. Certain epitopes, however, are ubiquitous to HBc/e proteins and these are probably exposed on the surface of HBc particles.

Functions of hepatitis B surface proteins.

HBV surface proteins play a number of functional roles in cellular infection, viral synthesis and in immune responses of the host. Three coterminal proteins of differing sizes and three subdomains of the individual molecules can be recognized. In this brief review, functions of the various proteins and domains are described and their significance as potential immunogens is discussed. Although it is apparent that the surface proteins are involved in the development of persistent HBV infections, the underlying mechanisms of liver involvement remain unknown.

Characterization of the endogenous protein kinase activity of the hepatitis B virus.

During the assembly of the nucleocapsid of the hepatitis B virus a protein kinase, probably of cellular origin, is encapsidated. This enzyme phosphorylates serine residue(s) localized within the lumen of the particle. By using purified, liver-derived core particles, we characterized the protein kinase activity in the presence of different ions and inhibitors. Controls were performed with cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) and recombinant core particles. We showed that the endogenous protein kinase of the core particles was not inhibited by H89, a specific inhibitor of PKA. Staurosporine, a selective inhibitor of PKC inhibited the endogenous kinase activity only within the first minutes of the reaction. In contrast, quercetine, a selective inhibitor of the protein kinase M (PKM) did not inhibit during the first minutes but inhibited efficiently during later phases of incubation. PKM represents an enzymatically active proteolytic fragment of PKC. These results suggest that PKC is encapsidated into human core particles and is converted to PKM during the in vitro reaction. This conclusion implies the association of a protease activity localized with the HBV nucleocapsid inside liver-derived core particles.

Assay of antibodies to hepatitis C virus protein C100-3 in blood donors from northern Germany.

The prevalence of anti-C100-3 increases with age from 0.41% to 1.26%. It is more frequent in donors with elevated ALT (4.5%). Most ALT elevations, however, are not related to anti-C100-3. Low EIA signals (< 3 x cutoff) are often non-specific. The cutoff value should be 2.5 times higher. High EIA signals correlate with ALT elevations.

Absence of free core antigen in anti-HBc negative viremic hepatitis B carriers.

Using enzyme immune assay and immune electron microscopy, we have examined the sera of immune-suppressed anti-HBc negative HBV-infected patients for the presence of HBcAg. Our results suggest that free HBV core particles are absent or present only in minute amounts in the blood of chronic carriers and that at the most, only minimal amounts of core antigen are found on the surface of the virus particles.

Detection of transcriptionally active hepatitis B virus DNA in peripheral mononuclear blood cells after infection during immunosuppressive chemotherapy using the polymerase chain reaction.

Using PCR we have studied mononuclear peripheral blood leucocytes (PMBLs) from HBV-infected immunosuppressed patients in order to detect the presence of HBV genomes. Our results indicate that non-transient PMBL infection is common in immunotolerant carriers. In addition, the presence of pregenomic mRNA sequences suggests that virus replication may take place in PMBLs, possibly implicating the latter as a source of virus after replication has ceased in the liver.

Clinical and immunological aspects of hepatitis B virus infection in children receiving multidrug cancer chemotherapy.

For two reasons hepatitis B virus infection is an important problem in patients with cancer. First, multidrug cancer chemotherapy may reactivate or worsen a previously benign chronic HBV infection. Second, patients undergoing cancer chemotherapy are at an increased risk of acquiring and spreading HBV which may result in an endemic infection. HBV reactivation may precipitate into a severe acute disease including fulminant hepatitis. In contrast, the acquisition of HBV during cancer chemotherapy commonly takes a mild clinical course but frequently leads to persistently high viremia. This state of immunotolerance to viral antigens allows viral replication without any sign of liver cell destruction. Withdrawal of chemotherapy does not cause significant changes if infection occurred during cytotoxic chemotherapy. Infection with HBV during cancer chemotherapy, therefore, may be considered as a model of an induced antigen-specific immunotolerance. In agreement with this hypothesis, vaccination against HBV during cancer chemotherapy does not prevent spread of HBV in oncology wards as it does not produce significant anti-HBs titers. Furthermore, vaccination even suppresses the immune response to later booster doses after chemotherapy has been withdrawn.

International collaborative study on the second EUROHEP HCV-RNA reference panel.

Eighty-six laboratories participated in a collaborative study and tested the second EUROHEP HCV-RNA reference panel. The coded panel comprised 4 HCV-RNA positive plasma samples (one weak positive), 6 HCV-RNA negative plasma samples and two dilution series of HCV-RNA genotype 1 and 3 plasma standards. The 86 laboratories submitted 136 coded data forms for evaluation. Of these data sets 99 were tested using a PCR assay developed in-house, 28 using a commercially available HCV-PCR test (AMPLICOR, Roche Diagnostic Systems) and 9 using other amplification methods. Twenty-two data forms (16%) had faultless results, 39 (29%) missed the weak positive sample only and 75 data sets (55%) had false positive and/or false negative results. Participants using the commercial HCV-PCR test tended to reach a sufficient quality score more often than investigators using assays developed in-house (64% versus 45%, P = 0.11). The UNG system in the commercial HCV-PCR test did not prevent five laboratories generating false-positive results in the 6 HCV-RNA negative samples. Among the laboratories with satisfactory results, up to 10000-fold differences in sensitivity were observed in the dilution series. The 50% and 90% laboratories detection endpoints in the dilution series of the HCV genotype 1 plasma standard were approximately 600 genome equivalents per ml (geq/ml) and 7750 geq/ml according to a standard applied in a signal amplification assay (bDNA, Chiron). Our results suggest that the detection efficiency for genotype 3 by commercial HCV-RNA assays is lower than by the in-house assays. Internationally characterized HCV-RNA plasma standards should be made available for validation and standardization of HCV-RNA assays for HCV diagnosis and virological safety testing of blood products.

Performance of hepatitis B surface antigen tests with the first WHO international hepatitis B virus genotype reference panel.

BACKGROUND: Standardization of hepatitis B surface antigen (HBsAg) tests is indispensable for consistent quality and comparability. Ideally, the assays should detect all known hepatitis B virus (HBV) genotypes equally well. OBJECTIVE: Development of an HBV genotype reference panel for HBsAg assays representing the most prevalent HBV subgenotypes to address commutability and traceability of the heat-inactivated 2nd WHO International Standard (IS) for HBsAg in relation to native HBsAg and to HBV genotypes. STUDY DESIGN: An HBV panel of 15 non-inactivated lyophilized specimens representing the subgenotypes A1, A2, B1, B2, C2, D1-D3, E, F2, and H was evaluated in parallel to the IS by 15 laboratories using 19 different HBsAg tests and tree unitages. The virus content of the samples was reduced by ultracentrifugation and dilution to <2×10(4) IU HBV DNA/mL. RESULTS: Twenty-two qualitative and 6 quantitative data sets were evaluated. Overall, the results demonstrated consistent detection of HBV genotypes by the majority of tests with a mean potency variability relative to the IS of 36%. Some assays showed significant genotype-dependent differences in analytical sensitivity. Some tests were more sensitive with the IS, others less. On average, one IU HBsAg corresponded to 0.88±0.20 ng HBsAg protein. CONCLUSIONS: The panel was accepted by the WHO as the "1st International Reference Panel for HBV genotypes for HBsAg-based assays". The panel is a helpful complementation to the IS to validate HBV genotype specific analytical test sensitivities.