Long‐Term Entecavir Treatment Results in Sustained Antiviral Efficacy and Prolonged Life Span in the Woodchuck Model of Chronic Hepatitis Infection

Colonno, Richard J.; Genovesi, Eugene V.; Medina, Ivette; Lamb, Lucinda; Durham, Stephen K.; Huang, Meei‐Li; Corey, Lawrence; Littlejohn, Margaret; Locarnini, S.; Tennant, Bud C.; Rose, Burt; Clark, Junius M.

doi:10.1086/324003

Cited by 132 publications

(85 citation statements)

References 33 publications

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“…It is particularly refractory to current antiviral therapy, yet its elimination is a prerequisite for any real cure of an HBV infection. Although the half-life of preformed CCC DNA remains a hotly debated issue (21,33,40,43,44,58,70), effective, long-term antiviral treatment to block the synthesis of RC DNA, thus depleting the precursor to CCC DNA, can lead to significant reduction of CCC DNA in vitro and in vivo (1,3,8,11,18,22,49,64,69). These results indicate that continuous replenishment of the CCC DNA pool, via either de novo infection or intracellular amplification, is required to maintain persistent infections.…”

Section: Discussionmentioning

confidence: 99%

Formation of Hepatitis B Virus Covalently Closed Circular DNA: Removal of Genome-Linked Protein

Gao

2007

J Virol

177

247

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Hepatitis B virus (HBV) contains a small, partially double-stranded, relaxed circular (RC) DNA genome. RC DNA needs to be converted to covalently closed circular (CCC) DNA, which serves as the template for all viral RNA transcription. As a first step toward understanding how CCC DNA is formed, we analyzed the viral and host factors that may be involved in CCC DNA formation, using transient and stable DNA transfections of HBV and the related avian hepadnavirus, duck hepatitis B virus (DHBV). Our results show that HBV CCC DNA formed in hepatoma cells was derived predominantly from RC DNA with a precise junction sequence. In contrast to that of DHBV, HBV CCC DNA formation in cultured cells was accompanied by the accumulation of a RC DNA species from which the covalently attached viral reverse transcriptase (RT) protein was removed (protein-free or PF-RC DNA). Furthermore, whereas envelope deficiency led to increased CCC DNA formation in DHBV, it resulted mainly in increased PF-RC, but not CCC, DNA in HBV, suggesting that the envelope protein(s) may negatively regulate a step in CCC DNA formation that precedes deproteination in both HBV and DHBV. Interestingly, PF-RC DNA, in contrast to RT-linked RC DNA, contained, almost exclusively, mature plus-strand DNA, suggesting that the RT protein was removed preferentially from mature RC DNA.Hepatitis B virus (HBV) infection is a global public health problem, with over 300 million chronically infected patients worldwide (16). Chronic HBV infection is associated with a high risk of developing severe liver diseases, including cirrhosis and hepatocellular carcinoma, and results in a million deaths annually. HBV is a member of the Hepadnaviridae, a family of small, hepatotropic DNA viruses (37), which also includes related animal viruses, such as duck hepatitis B virus (DHBV) (41) and woodchuck hepatitis virus (WHV) (63). All hepadnaviruses carry a small (ca. 3.2 kb), relaxed circular (RC), partially double-stranded DNA genome ( Fig. 1) and replicate through an RNA intermediate, the pregenomic RNA (pgRNA), by reverse transcription (41,51,54,59,60).A critical early step in the HBV infection process is the conversion of the genomic RC DNA, present in the virions, into a covalently closed circular (CCC) DNA, which is found in the host cell nucleus (26,39,47,65). Following entry into the host cells, the virion RC DNA is released into the nucleus for conversion into CCC DNA, which is the first viral product to be made in a newly infected cell. CCC DNA then serves as the viral transcriptional template for the synthesis of all viral RNAs by the host RNA polymerase II. Among the viral RNAs produced is the pgRNA, which is packaged together with the reverse transcriptase (RT) protein into immature nucleocapsids comprised of the viral capsid or core protein (4, 23). The pgRNA is then reverse transcribed within the nucleocapsids to make RC DNA by the multifunctional RT (27), using a reverse transcription pathway that is similar to, yet distinct from, that used by conventional retroviruses ...

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Section: Discussionmentioning

confidence: 99%

Formation of Hepatitis B Virus Covalently Closed Circular DNA: Removal of Genome-Linked Protein

Gao

2007

J Virol

177

247

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“…Recently, several nucleos(t)ide analogues such as lamivudine [Dienstag et al, 1995], adefovir dipivoxil [Chin et al, 2001], and entecavir [Colonno et al, 2001] have been found to consistently produce rapid and dramatic decreases in viremia [Dienstag et al, 1995[Dienstag et al, , 1999Lai et al, 1998;Suzuki et al, 1999]. For the serological monitoring of chronic hepatitis patients under treatment with nucleos(t)ide analogues, improvement of alanine transaminase level, seroconversion from HBe antigen (HBeAg)-positive to anti-HBe antibody (HBeAb)-positive, and peripheral HBV DNA concentration are used as markers in chronic active hepatitis, and both HBeAg seroconversion and HBV DNA levels below the detection limit and/or of 10 5 copies/ml are commonly used as primary treatment endpoints [Lok et al, 2004].…”

Section: Introductionmentioning

confidence: 99%

Correlation between serum hepatitis B virus core‐related antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients

Suzuki

Miyakoshi

Kobayashi

et al. 2008

Journal of Medical Virology

171

189

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Nucleos(t)ide analogues are utilized for the treatment of chronic HBV infection, and HBe seroconversion and HBV DNA levels are commonly used as markers of viral status and as primary treatment endpoints. Recently, a new assay was prepared for the detection of serum HBV corerelated antigen (HBcrAg), consisting of HBcAg, HBeAg, and p22cr, which is a precore protein from amino acid À28 to at least amino acid 150, by coding the precore/core region. In this study, we examined the correlation between serum HBcrAg concentration and viral status by the analysis of serum HBeAg, HBsAg, peripheral HBV DNA, and intrahepatic covalently closed circular DNA (cccDNA) in 57 chronic hepatitis B patients. Intrahepatic cccDNA was detected in all 57 patients, 42 patients were HBcrAg-positive, and serum HBcrAg concentration level was closely correlated with cccDNA. Additionally, positive HBcrAg concentration level results were observed in 6 out of 13 HBsAg seroclearance patients and 20 out of 31 HBV DNA-negative patients. Moreover, the correlation between HBcrAg and cccDNA in these 31 HBV DNAnegative patients was statistically significant (r ¼ 0.482, P ¼ 0.006). These data suggest that serum HBcrAg concentration is well correlated with intrahepatic cccDNA level, and that the measurement of serum HBcrAg may be clinically useful for monitoring intrahepatic HBV viral status, especially in patients under treatment with nucleos(t)ide analogues.

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“…More potent antivirals, like entecavir (4,9), and multiple vaccinations of more than three injections could further enhance specific immune responses and yield better results.…”

mentioning

confidence: 99%

Combination of an Antiviral Drug and Immunomodulation against Hepadnaviral Infection in the Woodchuck Model

Yao

et al. 2008

J Virol

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Approximately 400 million people worldwide are chronically infected with hepatitis B virus (HBV). Therapy with interferon or nucleoside/tide analogs is not satisfactory due to the low responder rate and resistance development (16,18,20,33). To date, immunotherapy against chronic hepatitis B has not yet achieved satisfactory results (7,14,17,31,32,41,44). Given the crucial role of cytotoxic T lymphocytes in the control of HBV infections, new therapeutic vaccines with the ability to stimulate vigorous, broad HBV-specific cytotoxic T lymphocyte responses are needed (3,11,26,38,39).Several studies of therapeutic vaccinations have been carried out in the woodchuck model (reviewed in references 24, 25, 34, and 35) and demonstrated collectively that B-or T-cell responses to viral antigens could be induced in chronic woodchuck hepatitis virus (WHV) carriers (12,13,23,30). Yet, none of these studies have demonstrated the capability of vaccines to suppress viral replication. Here, we carried out a proof-of-principle experiment using DNA vaccines alone and DNA vaccines combined with immune complexes (IC) in the woodchuck model. IC of HBsAg and anti-HBs have been tested in patients and in transgenic mice (42,43,45,46). IC are more efficiently taken up by antigen-presenting cells than free antigens, leading to an improved presentation to T cells. DNA vaccines are potent inducers of T-cell responses. They could stimulate HBV-specific immune responses in humans and prevent hepadnaviral infections in the animal model (21,22,37). In addition, woodchucks were pretreated with lamivudine, a potent antiviral drug against HBV with the ability to enhance T-cell responses in chronically HBV-infected patients (1, 2).A total of 10 chronically WHV-infected woodchucks (from Northeastern Wildlife, Ithaca, NY) were first treated with 15 mg of lamivudine daily and randomly divided into three groups: the control group (n ϭ 2), a group vaccinated with WHV surface antigen (WHsAg)-IC and DNA (n ϭ 4), and a group vaccinated with DNA (n ϭ 4). The immunization schedule is presented in Fig. 1.The DNA vaccines consisted of an equimolar mixture of three plasmids, pWHsIm, pWHcIm, and pWIFN, expressing WHsAg, WHV core antigen (WHcAg), and woodchuck gamma interferon, respectively, as described previously (21, 37). To produce IC for woodchucks, antibodies to WHs (antiWHs) and WHsAg were titrated by the checkerboard method to determine the stoichiometry of the antigen and antibodies. The appropriate concentration of WHsAg was chosen and incubated with antibodies at 37°C for 30 min and then incubated overnight at 4°C, resulting in a preparation with a final concentration of 80 g WHsAg/ml in complex with anti-WHs. The vaccine consisted of 20 g WHsAg-IC and 250 g plasmid pWHsIm DNA per 0.5-ml dose. The DNA vaccines and IC-DNA vaccines were administered by intramuscular injections (21,22).The following parameters were determined by the indicated methods: serum WHV DNA concentrations as genome equivalents (GE) by real-time PCR with the LightCycler DNA Master S...

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Long‐Term Entecavir Treatment Results in Sustained Antiviral Efficacy and Prolonged Life Span in the Woodchuck Model of Chronic Hepatitis Infection

Cited by 132 publications

References 33 publications

Formation of Hepatitis B Virus Covalently Closed Circular DNA: Removal of Genome-Linked Protein

Formation of Hepatitis B Virus Covalently Closed Circular DNA: Removal of Genome-Linked Protein

Correlation between serum hepatitis B virus core‐related antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients

Combination of an Antiviral Drug and Immunomodulation against Hepadnaviral Infection in the Woodchuck Model

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