N-terminal myristoylation-dependent masking of neutralizing epitopes in the preS1 attachment site of hepatitis B virus

The N-terminally myristoylated preS1 domain of the large hepatitis B surface protein (LHBs) mediates specific attachment of hepatitis B virus (HBV) to hepatocytes. Its B-cell epitopes leading to neutralization of infectivity are not yet characterized.We inserted C- and N-terminal preS1 peptides into the most immunogenic region of HBV core particles, therewith immunized Balb/c mice and determined binding properties and neutralization potential of resulting antibodies in vitro.The particles with preS1 inserts were highly immunogenic and the corresponding anti-preS antibodies strongly bound to HBV particles from chronic carriers infected with different HBV genotypes A-F. However, antibodies bi…

Hepatic expression patterns of the large and middle hepatitis B virus surface proteins in viremic and nonviremic chronic hepatitis B.

The envelope of hepatitis B virus consists of large, middle, and small hepatitis B surface proteins. Recent data from in vitro studies suggest that intracellular expression and distribution of the three polypeptides may be variable. These observations in artificial expression systems prompted this analysis of the occurrence and distribution of the three hepatitis B surface proteins in the liver tissue of substantial viremic (hepatitis B virus DNA- and hepatitis B e antigen-positive) and low-viremic or nonviremic (hepatitis B virus DNA-negative, anti-hepatitis B e antigen-positive) carriers by specific monoclonal antibodies against large, middle, and small proteins. Patients with an active f…

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 particl…

Assay of hepatitis B virus DNA by polymerase chain reaction and its relationship to Pre-S- and S-encoded viral surface antigens

The polymerase chain reaction was evaluated as a diagnostic tool in 72 chronic hepatitis B virus carriers. Hepatitis B virus DNA was detectable in the serum of HBsAg—positive virus carriers using aliquots as small as 100 al. The detection limit for cloned hepatitis B virus DNA was 100 ag. Primer pairs for different regions of the HBV genome resulted in different sensitivity. Detection of the amplified hepatitis B virus DNA by Southern blotting and subsequent scintillation counting or densitometry allowed a semiquantitative assay. Using several primer pairs in parallel for optimal detection, all HBeAg-positive HBsAg carriers, 80% of HBe antibody—positive symptomatic HBsAg carriers and 57% of…

Immune blot analysis of viral surface proteins in serum and liver of patients with chronic hepatitis B virus infection

The small and the middle surface proteins of hepatitis virus form either the virion or the 22 nm particle both of which are secreted. The large surface protein by itself remains cell bound in artificially transfected cell culture unless it is accompanied by an excess of the smaller protens. Its behavior in vivo is not yet well studied. Using specific monoclonal antibodies for immunoblotting, we found an abundance of small surface protein in the serum of chronic virus carriers and moderate amounts in the liver irrespective of viremia. The large surface protein was present in the serum and the liver of viremic carriers. In nonviremic carriers, the large protein was absent from serum, but in t…

Analysis of the precore DNA sequence and detection of precore antigen in liver specimens from patients with anti-hepatitis b e—positive chronic hepatitis

A number of naturally occurring hepatitis B virus (HBV) mutants unable to synthesize the hepatitis B e antigen (HBeAg) have been identified in patients characterized by HBV DNA and anti-HBe in their serum. Because the analysis of the HBV-associated DNA and antigens in the liver tissue is still not complete, we investigated the precore sequence of HBV DNA and its encoded proteins in the liver tissue of 32 patients positive for HBV DNA and anti-HBe in their serum. Three different groups of patients were identified. Group I (n = 14) was characterized by viral DNA sequences with a G-A transition in the distal precore gene region, thus creating a termination codon (TAG). Liver tissue from this g…

Statements from the Taormina expert meeting on occult hepatitis B virus infection

Giovanni Raimondo*, Jean-Pierre Allain, Maurizia R. Brunetto, Marie-Annick Buendia, Ding-Shinn Chen, Massimo Colombo, Antonio Craxi, Francesco Donato, Carlo Ferrari, Giovanni B. Gaeta, Wolfram H. Gerlich, Massimo Levrero, Stephen Locarnini, Thomas Michalak, Mario U. Mondelli, Jean-Michel Pawlotsky, Teresa Pollicino, Daniele Prati, Massimo Puoti, Didier Samuel, Daniel Shouval, Antonina Smedile, Giovanni Squadrito, Christian Trepo, Erica Villa, Hans Will, Alessandro R. Zanetti, Fabien Zoulim

Treatment of hepatitis B surface antigen (HBsAg)-positive chronic hepatitis with recombinant leucocyte α-A interferon

A total of 32 individuals with HBsAg-positive and anti-delta-negative chronic hepatitis were treated with recombinant alpha-A interferon in phase I and phase II studies. In 5/32 patients HBsAg could be eliminated and in 19/32 individuals HBeAg became negative including all those who also eliminated HBsAg. Side-effects were tolerable in most patients and were readily reversible upon discontinuation of interferon therapy. In conclusion, treatment of HBsAg-positive chronic hepatitis with interferon seems to be a promising therapeutic approach. Future studies will have to establish the optimal dose, duration of treatment and factors predicting a favourable outcome of the treatment.

Untersuchungen zum Verteilungsmuster von Prä S und S-kodierten viralen Hüllproteinen des Hepatitis B Virus (HBV) auf molekularer Ebene im Serum und Lebergewebe von chronischen HBsAg-Trägern

Die biologische Bedeutung der PraS-kodierten Hepatitis B Virus (HBV) — Hullproteine — insbesondere ihre Regulation innerhalb der Leberzelle — ist noch unklar. Daher war es Ziel unserer Untersuchungen, die PraS- und S-kodierten Hullproteine vergleichend im Serum und im Lebergewebe auf molekularer Ebene zu untersuchen.

Biological standards for hepatitis B virus assays.

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 si…

Fine-mapping of the B-cell epitope domain at the N-terminus of the preS2 region of the hepatitis B surface antigen

In this study, we report the exact localization and substitutional characterization of a B-cell epitope domain at the N-terminus of the preS2 region of the hepatitis B surface antigen. A set of deletion variants containing preS2 sequences of different length was generated on the basis of frCP as a carrier. It was found after Western blot analysis that three monoclonal antibodies (MAbs) (2-11B1, 3-11C2, HB.OT10) recognized the linear preS2 sequence within the amino acid (aa) stretch 3-WNSTTFHQTLQDP-13. The importance of each aa residue of the epitope was proved by comparison of antibody binding to alanine-substituted peptides in both free-peptide and Pepscan variants.