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Biomedical subjects

Stephen A Locarnini

Publications and source records attributed to Stephen A Locarnini.

14 recordsLinked to original sources
PubMed2007

In vitro study of the effects of precore and lamivudine-resistant mutations on hepatitis B virus replication.

Understanding the consequences of mutation in the hepatitis B virus (HBV) genome on HBV replication is critical for treating chronic HBV infection. In this study, HBV replication in HepG2 cells initiated by transduction with precore (PC), rtM204I, and wild-type (wt) HBV recombinant baculoviruses was compared. The pattern and magnitude of HBV replication initiated by the PC HBV recombinant baculovirus were similar to those observed for wt HBV throughout the time course examined. In contrast, when the rtM204I mutation was introduced into wt HBV, by day 10 postinfection the levels of intra- and extracellular HBV DNA were markedly reduced compared to those for wt HBV. Although the rtM204I mutation reduced the production of HBV replicative intermediates, no effect on the level of covalently closed circular DNA or HBV transcripts was observed at late time points. Coinfection studies with different ratios of wt and rtM204I baculoviruses showed that the rtM204I variant did not produce a product that inhibited HBV replication. However, the combination of the wt and rtM204I baculoviruses yielded HBV DNA levels at late time points that were greater than those for the wt alone, suggesting that wt polymerase may function in trans to boost rtM204I replication. We concluded that the rtM204I mutation generates a polymerase that is not only resistant to lamivudine but also replicates nucleic acids to lower levels in vitro.

Animals↗
PubMed2006

SeqHepB: a sequence analysis program and relational database system for chronic hepatitis B.

SeqHepB is a combination of a HBV genome sequence analysis program and a relational database that houses data collected from multiple data sources. Registered users can access the sequence analysis component of SeqHepB online for rapid and detailed interrogation of HBV genomic sequences. Its main function is to determine the HBV genotype, identify key mutations associated with antiviral resistance, and identify clinically important HBV mutants. All information generated is uploaded into a database and integrated with patient medical records, pathology laboratory tests, and supplemental virology results such as in vitro drug cross-resistance values. Combined with structured query language (SQL) queries developed in the database, it is possible to extract and correlate clinical, virological, and in vitro phenotypic data rapidly and efficiently. An important component of SeqHepB is its ability to integrate mutations detected within the reverse transcriptase (RT) and locate them onto a three-dimensional (3D) model of the HBV RT that can be viewed at any angle with known antiviral drug molecules in the catalytic pocket of the enzyme. SeqHepB will enable virologists and physicians to individualise patient management, cope with the explosion of antiviral associated HBV mutations, and to conduct cross-sectional retrospective or prospective studies on HBV-infected individuals during therapy.

DNA Mutational Analysis↗
PubMed2006

Chronic hepatitis B: current testing strategies.

The worldwide burden of hepatitis B mandates accurate and timely diagnosis of patients infected with the hepatitis B virus (HBV) and the use of treatment strategies derived from evidence-based guidelines. HBV is a DNA virus that produces a series of viral protein products circulating HBV DNA. Serologic and nucleic acid testing are critical to disease prevention and treatment objectives. Information from such testing helps determine patients' infectivity and immune status, appropriate monitoring strategies, and the efficacy of treatment, as well as providing data that contributes to a better understanding of the natural history and epidemiology of the disease. This article reviews the clinical use of state-of-the-art serologic and nucleic acid tests, including the relevance of hepatitis B e antigen and antibody and HBV DNA measurements as markers of disease activity. Viral load can be used to distinguish between active and inactive disease, define response to therapy, and detect the development of antiviral resistance. Some recent reports have suggested that high viral load is associated with poorer patient outcomes (eg, more rapid progression to cirrhosis and a higher incidence of hepatocellular carcinoma). Durable suppression of HBV DNA is evolving to become the primary goal of therapy, although all currently licensed medications have used histology as the primary end point of therapy. Suggested frequencies for HBV DNA monitoring are presented.

DNA, Viral↗
PubMed2006

Antiviral drug resistance: clinical consequences and molecular aspects.

Antiviral drug resistance now poses a major problem for the management of patients with chronic hepatitis B. In theory, resistance may be prevented if a sufficiently potent antiviral drug, or combination of antiviral agents, is used that prevents viral replication and thereby the ongoing selection of hepatitis B virus quasispecies. Emergence of drug resistance in patients with hepatitis B generally results in progression of liver disease and in some cases, significant clinical deterioration if hepatic reserve is compromised. Currently, there are two major patterns of resistance mutations found in the viral reverse transcriptase (rt) that can be selected during monotherapy: (1) those that include the codon rtM204, which is part of the catalytic domain (YMDD) of the enzyme; (2) and those that do not include the codon. The rtM204I/V is selected by lamivudine and L-nucleosides. It is also part of the entecavir resistance profile and the tenofovir-lamivudine combination resistance profile. In contrast, resistance to adefovir is associated with mutations at rtN236T +/- rtA181V. A reasonable clinical goal is to develop an overall strategy that prevents the selection of resistance. These strategies have yet to be optimized for hepatitis B, but may include multiple therapies such as immune-based therapies in combination with one or more nucleoside analogue treatments. Future treatment protocols can be modeled on the use of multiple agents comprising highly active anti-retroviral therapy regimens that have been developed for the successful management of patients infected with human immunodeficiency virus.

Antiviral Agents↗
PubMed2004

Hepatic failure due to fibrosing cholestatic hepatitis in a patient with pre-surface mutant hepatitis B virus and mixed connective tissue disease treated with prednisolone and chloroquine.

Fibrosing cholestatic hepatitis (FCH) is a severe variant of hepatitis B infection that has until recently been described almost exclusively in the setting of organ transplantation and HIV infection. This case report describes a patient with pre-surface (pre-S) mutant hepatitis B virus (HBV) infection who developed a fatal form of FCH after high dose prednisolone for mixed connective tissue disease (MCTD). The role of corticosteroids and pre-S viral mutation in the pathogenesis of the disease is discussed, and the importance of early diagnosis is emphasised. This report alerts the physician to the need for close monitoring of LFTs and HBV DNA of hepatitis B carriers during immunosuppressive therapy regardless of the indication. As in the transplantation setting, viral DNA levels should be kept to undetectable if viral replication or recurrence is to be prevented.

Adult↗
PubMed2003

Effects of interferon alpha therapy on the catalytic domains of the polymerase gene and basal core promoter, precore and core regions of hepatitis B virus.

AIMS: The aim of the present study was to examine the catalytic domains of the polymerase gene, the basal core promoter and the precore and core regions of the hepatitis B virus (HBV) genome for specific mutations. These may account for the response to interferon alpha (IFN-alpha) treatment, which may have prognostic value. METHODS: Multiple serum samples were collected prospectively from 30 patients with chronic active hepatitis B who were treated with IFN-alpha. Patients were assigned to one of three groups: group A (n = 11) and group B (n = 10) individuals were hepatitis B e antigen (HBeAg)-positive prior to treatment. Group A patients underwent HBeAg seroconversion after treatment while group B patients did not. Group C (n = 9) patients were HBeAg-negative prior to treatment. The HBV DNA was extracted from the sera collected before, during and after treatment and the various genomic regions were amplified, sequenced and examined for mutations. RESULTS: During IFN-alpha therapy, multiple changes were found in the catalytic domains of the HBV polymerase gene in all groups. The frequency of mutations and associated amino acid changes were highest in virus from group C patients and lowest in group A patients. The interdomain regions of the viral polymerase were the most affected. Multiple mutations were also found in the precore, core and core promoter regions. However, no specific mutations were associated with clinical response or outcome. CONCLUSIONS: During IFN-alpha treatment, multiple mutations occurred in the HBV genome, including the catalytic domains of the polymerase gene. Changes that did occur could not be correlated to the clinical response or treatment outcome. However, no mutations were found that have been linked to lamivudine escape, indicating that lamivudine therapy would be effective in IFN-alpha non-responder patients.

Adult↗
PubMed2003

Effect of the G1896A precore mutation on drug sensitivity and replication yield of lamivudine-resistant HBV in vitro.

Hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) is frequently caused by a mutation (G1896A) in the hepatitis B virus (HBV) precore (PC) reading frame that creates a stop codon, causing premature termination of the PC protein. During lamivudine treatment, drug resistance develops at a similar rate in HBeAg positive and HBeAg negative CHB. Lamivudine-resistant HBV mutants have been shown to replicate inefficiently in vitro in the absence of PC mutations, but it is unknown whether the presence of PC mutations affects replication efficiency or antiviral sensitivity. This study utilized the recombinant HBV baculovirus system to address these issues. HBV baculoviruses encoding the G1896A PC stop codon mutation were generated in wild-type (WT) and lamivudine-resistant (rtM204I and rtL180M + rtM204V) backgrounds, resulting in a panel of 6 related recombinant baculoviruses. In vitro assays were performed to compare the sensitivities of the PC mutant viruses with lamivudine and adefovir and to compare relative replication yields. The PC mutation did not significantly affect sensitivities to either adefovir or lamivudine. WT HBV and PC mutant HBV showed similar replication yields, whereas the replication yields of the lamivudine-resistant mutants were greatly reduced in HBeAg positive HBVs, confirming previous observations. However, the presence of the PC mutation was found to compensate for the replication deficiency in each of the lamivudine-resistant mutants, increasing the replication yields of each virus. In conclusion, the PC stop codon mutation appears to increase the replication efficacy of lamivudine-resistant virus but does not affect in vitro drug sensitivity.

Adenine↗
PubMed2002

Restoration of replication phenotype of lamivudine-resistant hepatitis B virus mutants by compensatory changes in the "fingers" subdomain of the viral polymerase selected as a consequence of mutations in the overlapping S gene.

The introduction of lamivudine (LMV) for the treatment of chronic hepatitis B infection has been an important advance in the management of this disease. However, the long-term efficacy of LMV may become limited by the emergence of antiviral-resistant hepatitis B virus (HBV) mutants. The two most common LMV-resistant mutants produce changes in the viral polymerase protein (rt) of rtM204I and rtL180M/M204V (previously rtM550I and rtL526M/M550V). A number of studies have demonstrated that these HBV mutants appear to be replication impaired, both in vitro and in vivo. The detection and selection of compensatory mutations in the polymerase protein that restore the replication phenotype of these HBV mutants have been poorly described to date. The effects of mutations in the fingers subdomain of the viral polymerase protein arising as a consequence of vaccine and hepatitis B immune globulin (HBIg) selected changes in the overlapping envelope gene (S), and a determinant of the hepatitis Bs antigen (HBsAg) were analyzed in vitro. The LMV-resistant HBV mutants rtM204I and rtL180M/M204V produced substantially weaker HBV DNA replicative intermediate signals by Southern blot analysis and less total intracellular HBV DNA by real-time PCR compared to wild-type virus. The viral polymerase protein of these mutants produced little detectable radiolabeled HBV DNA in an endogenous polymerase assay. In contrast, the HBV a determinant HBIg/vaccine escape mutants sP120T, sT123N, sG145R, and sD144E/G145R (that produce rtT128N, Q130P, rtW153Q, and rtG153E respectively) yielded as much virus as wild-type HBV while the sM133L (rtY141S) mutant was replication impaired. Two of these mutants, rtT128N and rtW153Q, when introduced into a replication-competent HBV vector containing the rtL180M/M204V polymerase mutation restored the replication phenotype of this LMV-resistant mutant. These viruses produced levels of intracellular HBV DNA as determined by Southern blot and real-time PCR that were comparable to those of wild-type HBV, indicating that the changes in the fingers subdomain were able to compensate for the reduced replication of the LMV-resistant mutations. Since these viruses carry mutations in the a determinant of HBsAg that may potentially decrease the ability of anti-HBs antibody to neutralize these viruses, these HBV mutants also have the potential to behave as vaccine escape mutants.

Cell Line↗
PubMed2002

Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy.

The prevalence of hepatitis B virus vaccine escape mutants has increased as a consequence of the introduction of global vaccination programs. Furthermore and as a consequence of the organization of the genome of hepatitis B virus (HBV) into overlapping reading frames, the selection of polymerase mutants during long-term lamivudine therapy can select viruses with changes in the overlapping S gene coding for the hepatitis B small antigen (HBsAg). We have investigated the role of lamivudine in selecting HBV mutants with antigenically altered HBsAg protein using pooled human vaccine sera in enzyme immunosorbent assays and radioimmunoassays. HBsAg proteins containing the vaccine escape mutations G145R and D144E/G145R demonstrated markedly reduced binding to anti-HBs antibody. HBsAg mutants including E164D, W196S, I195M, M198I, and E164D/I195M (corresponding to the polymerase protein changes of V519L, M550I, L526M/M550V V553I, and V519L/L526M/M550V) selected during lamivudine treatment also demonstrated reduced binding to anti-HBs antibody. These findings raise the possibility of lamivudine-resistant mutants arising that possess antigenically distinct HBsAg proteins.

DNA-Directed DNA Polymerase↗
PubMed2002

Clinical relevance of viral dynamics and genotypes in hepatitis B virus.

Advances in molecular diagnostic technology make it possible to accurately measure viral loads and this has allowed the detailed study of viral dynamics of HIV, hepatitis C virus (HCV) and hepatitis B virus (HBV). Following antiviral therapy, there are at least two phases of viral load decay: one corresponding to clearance of free virions and a second, slower phase corresponding to eradication of infected cells. Application of mathematical models allows for the assessment of antiviral efficacy and improved design of therapeutic regimens. The clinical application of these tools should help optimize patient outcome. Another advantage of molecular diagnostics is characterization of the heterogeneity of viruses in particular patient populations under selective pressure situations. The HBV can be classified into seven major genotypes (A-G) that have mainly a geographic distribution. Recent genotypic studies have revealed the clinical and therapeutic relevance of viral genotyping in HBV infections.

Antiviral Agents↗
PubMed2002

Mechanisms of drug resistance and novel approaches to therapy for chronic hepatitis C.

Hepatitis C virus (HCV) is now the major cause of transfusion-associated and parenterally transmitted viral hepatitis and accounts for a significant proportion of hepatitis cases worldwide. The majority of infections become persistent and approximately 20% of chronically infected individuals develop cirrhosis, which is strongly associated with progression to hepatocellular carcinoma. Molecular biological investigations into the structure and function of HCV and its genes has led to the identification of a number of potential targets for selective antiviral intervention. The present review summarizes current research activity into these novel drug targets and addresses the basis for clinical non-response in the current interferon-alpha-based therapies. Future therapeutic strategies that utilize HCV-specific antiviral agents should prove effective in controlling active viral replication, but the risk of emergence of drug-resistance will need to be addressed due to the quasispecies feature of HCV replication.

Antiviral Agents↗
PubMed2002

Advances in hepatitis C: what is coming in the next 5 years?

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Numerous advances have been made in the understanding of HCV replication, including detailed molecular characterization of its viral proteins and genomic RNA. The inability to grow HCV in cell culture had impeded the development of antiviral agents against this virus. To overcome this obstacle, a number of unique tools have been prepared, such as molecular clones that are infectious in the chimpanzee animal model of infection, and the development of a subgenomic replicon system in Huh7 cells. In addition, the major non-structural proteins have been crystallized, thus enabling rational drug design directed to these targets. Current developments in antiviral agents are reviewed in the context of these potential new viral targets for the future treatment of HCV in chronically infected individuals.

Antiviral Agents↗
PubMed2002

Evolving therapies for the treatment of chronic hepatitis B virus infection.

Despite the availability of prophylactic vaccines lamivudine and IFN-alpha, chronic hepatitis B remains an enormous global health problem. Several promising nucleosides/nucleotides are undergoing clinical trials, including adefovir dipivoxil, the latter of which is active against lamivudine-resistant hepatitis B virus (HBV). In addition to nucleosides/nucleotides, it will be important to develop new agents with different modes of action. Novel small molecule inhibitors, as well as gene therapy approaches, have produced encouraging results in vitro and in animal models. Additional immunomodulatory therapies, including thymosin-alpha 1, IL-12 and several therapeutic vaccines, are also being explored. Combination therapy with multiple nucleosides/nucleotides and other agents will play an important role in the treatment of hepatitis and may help achieve complete viral suppression, host-mediated elimination of infected cells and lasting immunity.

Adjuvants, Immunologic↗