Predictive value of aminotransferase and hepatitis B virus DNA levels on response to interferon therapy for chronic hepatitis B.

In a previously reported randomized controlled trial of interferon-alpha (IFN-alpha) for chronic hepatitis B, we found a significant difference in response between Chinese adults with elevated vs normal pretreatment aminotransferase (ALT) levels. The aim of this study was to determine the correlation between serum hepatitis B virus (HBV) DNA levels and response to IFN therapy. HBV DNA levels in residual stored sera from patients who participated in the above trial were quantified by a branched DNA (bDNA) assay. Nominal logistic regression was used to estimate the probability of response to IFN treatment as a function of pretreatment ALT and/or HBV DNA levels. We found a significant (P < 0.01) correlation between the HBV DNA levels at midtreatment and response to IFN therapy. Response was achieved in 53% of patients who had undetectable HBV DNA levels at midtreatment but in only 17% of those who remained HBV DNA positive (P < 0.01). In contrast, the probabilities of response for patients with baseline HBV DNA levels over the range 10 to 10000 million equivalents (MEq) ml-1 were almost identical. We also found a significant correlation between the pretreatment ALT levels and response to IFN therapy. The probabilities of response for patients with pretreatment ALT levels of 500 and 100 IU l-1 were higher than for patients with normal ALT levels by two and onefold, respectively. Our findings may help to improve the cost-effectiveness of IFN therapy for chronic hepatitis B by guiding the selection of patients for therapy and in optimizing the duration of treatment for the individual patient.

Hepatitis B virus S mutants in liver transplant recipients who were reinfected despite hepatitis B immune globulin prophylaxis.

Long-term hepatitis B immune globulin (HBIG) has been shown to reduce hepatitis B virus (HBV) reinfection in patients transplanted for hepatitis B. The aim of this study was to determine the prevalence of HBV S gene mutations in liver transplant recipients who developed recurrent hepatitis B despite HBIG prophylaxis, and to determine if these mutations can revert after withdrawal of HBIG. The entire S gene sequences in pre- and posttransplant sera from 20 patients who developed recurrent hepatitis B despite HBIG prophylaxis were compared. Ten (50%) patients had 18 amino acid substitutions involving the 'a' determinant in the posttransplant samples. These mutations were absent in 93% of the pretransplantation clones analyzed. There was a significant correlation between the development of mutations in the 'a' determinant region and the duration of HBIG therapy. Most of the mutations result in changes in predicted antigenicity of the S protein. During follow-up, mutations in 14 (78%) of 18 affected codons in the 'a' determinant region reverted back to the pretransplantation sequences; only 1 codon had a de novo change after the withdrawal of HBIG. Two control patients who did not receive HBIG had no change in the 'a' determinant in their posttransplantation samples. These data support the hypothesis that mutations in the S gene were induced or selected by immune pressure exerted by HBIG. HBV S mutants may play a role in HBV reinfection in liver transplant recipients who received HBIG prophylaxis.

High degree of conservation in the hepatitis B virus core gene during the immune tolerant phase in perinatally acquired chronic hepatitis B virus infection.

BACKGROUND: Mutations in the hepatitis B virus genome have been implicated in the persistence of hepatitis B virus infection and the pathogenesis of hepatitis B virus related liver disease. In view of the heterogeneity in published sequences, data from cross-sectional studies of unrelated subjects cannot differentiate true mutations from infections with variant sequences. AIMS/METHODS: We compared the hepatitis B virus core gene sequences of 42 HBsAg positive subjects from 11 Chinese families with those of the index patients (maternal carriers) to determine the frequency and rate of true hepatitis B virus core gene mutations in patients with chronic hepatitis B virus infection. RESULTS: Completely identical nucleotide sequences were present in all the family members and index patients in two families, suggesting that the hepatitis B virus core gene can be conserved for more than 20 years. The high degree of sequence conservation in these families is related to the young age of the subjects (mean 19.2+/-8.9 years), the fact that they were all HBeAg positive and that 75% of them had persistently normal aminotransferase levels. Longitudinal studies confirmed that mutations were rare in those who remained HBeAg positive with normal aminotransferase levels (immune tolerant phase), but significantly more common in HBeAg positive subjects who had elevated aminotransferase levels and in those who cleared HBeAg (immune clearance phase), the rates of nucleotide and amino acid changes were respectively: 0.28+/-0.12 vs 1.30+/-0.26/10(3) nt position/yr and 0.04+/-0.01 vs 0.18+/-0.5/10(2) codon/yr. CONCLUSIONS: Identical nucleotide differences could be found in the sequences of all the subjects in some families. These differences were more likely to be due to intra-familial transmission of stable variants. Sequence analysis based on comparisons with published sequences would have led to over-reporting of mutations. The hepatitis B virus core gene can remain highly conserved for more than two decades during the immune tolerant phase of perinatally acquired chronic hepatitis B virus infection. However, significant changes can occur within 2-3 years during the immune clearance phase.

High rate of mutations in the hepatitis B core gene during the immune clearance phase of chronic hepatitis B virus infection.

Cross-sectional studies reported that hepatitis B core gene mutations are associated with active liver disease and responsiveness to interferon therapy. In view of the heterogeneity in published sequences, it is not possible to tell whether the differences in sequences observed were true mutations that developed during the course of infection. We conducted a longitudinal study to determine the rate of hepatitis B core gene mutations and the timing of these mutations in relation to hepatitis B virus replication, activity of liver disease, hepatitis B virus replication, activity of liver disease, hepatitis B e antigen (HBeAg) seroconversion, and interferon therapy. Serial sera from 55 patients with chronic hepatitis B infection were analyzed by direct sequencing of the hepatitis B precore/core gene to identify the nucleotide and amino acid changes that emerged during during follow-up. Patients who remained HBeAg positive and had normal amino transferase levels (Group I) maintained higher serum hepatitis B virus (HBV) DNA levels but significantly lower rates of both nucleotide and amino acid changes during follow-up compared with patients who remained HBeAg positive but had elevated aminotransferase levels (Groups II) and patients who cleard HBeAg (Group III). The rates of nucleotide and amino acid changes in Groups I, II, III patients were: 0.4 +/- 0.1, 1.9 +/- 0.3, and 2.4 +/- 0.4/nucleotide position/year; and 0.04 +/- 0.02, 0.21 +/- 0.05, and 0.38 +/- 0.07/codon/year, respectively. Most of the amino acid changes in Groups II and III patients occured during or shortly after flares in amino transferase levels, before HBeAg seroconversion. Similar rates of nucleotide and amino acid changes were found in interferon treated versus untreated patients, and in responders versus nonresponders. There was no difference in the location or nature of the hepatitis B core gene mutations between patients with and without HBeAg seroconversion, interferon treated versus untreated patients, and interferon responders versus nonresponders. In summary, changes in the hepatitis B core gene sequence was rarely detected in patients who were still in the immune tolerant phase but very high rates of changes were found during the immune clearance of chronic hepatitis B infection. Interferon therapy did not induce a higher rate or specific pattern of mutations in the hepatitis B core gene. Response to interferon therapy in HBeAg positive patients was unrelated to the number or location of hepatitis B core gene mutations.

High level expression and phosphorylation of hepatitis B virus polymerase in insect cells with recombinant baculoviruses.

The hepatitis B virus polymerase open reading frame, as well as various subdomains of polymerase, was expressed in insect cells using the recombinant baculovirus expression system. Full-length polymerase was expressed at very low levels in a Spodoptera frugiperda cell line, the amino-terminal domain of polymerase was expressed at high levels, and other constructs were expressed at intermediate levels. Infections of a Trichoplusia ni cell line with the same recombinant baculoviruses resulted in high levels of protein production for all polymerase constructs. Each of the polymerase polypeptides was phosphorylated in insect cells. Since polypeptides with non-overlapping sequences were phosphorylated, polymerase must be phosphorylated at a minimum of two sites.