Licensed recombinant hepatitis B vaccines protect chimpanzees against infection with the prototype surface gene mutant of hepatitis B virus

Ogata, Norio; Côté, Paul J.; Zanetti, Alessandro; Miller, R.H.; Shapiro, Max; Gerin, John L.; Purcell, Robert H.

doi:10.1002/hep.510300309

Cited by 107 publications

(52 citation statements)

References 84 publications

(162 reference statements)

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“…The clinically relevant S-escape, precore stop codon, and BCP mutations all consist of G-A mismatches. [24][25][26][27][28][29] However, Tm shifts can also occur as a result of natural polymorphisms within the probe hybridization region of different HBV genotypes. Thus, according to the principle involved, the mismatches associated with natural polymorphism would result in larger Tm shifts than the single base-pair mismatches associated with the identified mutant, thereby allowing the mutant to be further distinguished from the wild-type virus.…”

Section: Discussionmentioning

confidence: 99%

Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

2002

Hepatology

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Current methods of detecting hepatitis B virus (HBV) mutations are time consuming, labor intensive, and not suitable for screening large numbers of samples. In the present study, we documented the advantages of a system that exploits differences in thermal stability between perfect match and mismatch hybrids, and thereby distinguishes between wild-type and mutants. Hybridization probes were designed complementary to specific wild-type HBV sequences in surface (S), precore, and basal core promoter (BCP) regions of the HBV genome (nt 587, 1896, and 1762/1764, respectively). Two probes were designed for each mutation: anchor probes were 3 labeled with fluorescein and sensor probes, 5 labeled with LC-Red 640, and 3 phosphorylated. Temperatures for each probe melted from amplification products were then determined in a melting program. Sera from 12 patients, each containing identified HBV mutants (6 S-escape, 1 precore, 1 BCP, and 4 mixed precore and BCP), and 5 control sera from patients with wild-type virus were analyzed. Genomic sequences of mutant and wild-type viruses were confirmed by direct sequencing. Real-time polymerase chain reaction (PCR) with fluorescent hybridization probes accurately identified each mutant and wild-type genome. Melting temperatures obtained from probe-product duplexes for the 3 mutants were distinguished from wild-type (>4.0°C, minimal) within 45 minutes. The sensitivity of the system was 100 copies/mL and as few as 5% of mutant among wild-type virus were detected. In conclusion, real-time PCR with fluorescent hybridization probes is a specific, sensitive, quantitative, and rapid means of detecting clinically relevant HBV mutants. (HEPATOLOGY 2002;36:723-728.) T he hepatitis B virus (HBV) is one of the most common infectious agents in humans and a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. [1][2][3] It is a partially double-stranded DNA molecule virus that replicates through an RNA intermediate by reverse transcription. 4,5 The reverse transcriptase enzyme responsible for this aspect of the replication cycle lacks proofreading function and, therefore, mutations to the genome are common. Indeed, it is now recognized that the majority of chronic HBV infections are associated with emergence of mutations throughout the viral genome. Some of these mutations result in the generation of diverse viral populations and different clinical outcomes. Those in the surface, core, precore, and basal core promoter (BCP) regions appear to have the greatest clinical relevance. 6,7 Additional mutations have also been described after therapeutic interventions such as antiviral therapy and vaccination. 8,9 Thus, the ability to detect these mutants is essential to further our understanding of the natural history of HBV and its response to antiviral therapy and immunoprophylaxis.Presently, the detection of HBV mutants is largely performed by restriction fragment length polymorphism analysis, 10,11 gap ligase chain reaction assay and/or direct sequencing. 12,13 These methods ...

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Section: Discussionmentioning

confidence: 99%

Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

2002

Hepatology

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“…The narrow host range of HBV and the lack of both in vitro systems and of convenient animal models have greatly hampered our understanding of the complete virus life cycle, as well as the development of more effective antiviral drugs aimed at eradicating the virus from chronic carriers. 3 Chimpanzees are the only animal species infectable with HBV, 4,5 but studies with these animals and evaluation of antiviral therapies are severely restricted because of their limited availability and high costs. Animal models based on HBV-related hepadnaviruses, such as woodchuck and Pekin duck hepatitis B viruses, are often used for assessment of antiviral drugs [6][7][8] and have provided important information about factors involved in establishment of virus infection, viral persistence, and hepatocarcinogenesis.…”

mentioning

confidence: 99%

Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus

et al. 2001

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Mice containing livers repopulated with human hepatocytes would provide excellent in vivo models for studies on human liver diseases and hepatotropic viruses, for which no permissive cell lines exist. Here, we report partial repopulation of the liver of immunodeficient urokinase-type plasminogen activator (uPA)/recombinant activation gene-2 (RAG-2) mice with normal human hepatocytes isolated from the adult liver. In the transplanted mice, the production of human albumin was demonstrated, indicating that human hepatocytes remained functional in the mouse liver for at least 2 months after transplantation. Inoculation of transplanted mice with human hepatitis B virus (HBV) led to the establishment of productive HBV infection. According to human-specific genomic DNA analysis and immunostaining of cryostat liver sections, human hepatocytes were estimated to constitute up to 15% of the uPA/RAG-2 mouse liver. This is proof that normal human hepatocytes can integrate into the mouse hepatic parenchyma, undergo multiple cell divisions, and remain permissive for a human hepatotropic virus in a xenogenic liver. This system will provide new opportunities for studies on etiology and therapy of viral and nonviral human liver diseases, as well as on hepatocyte biology and hepatocellular transplantation. Persistent infection with hepatitis B virus (HBV) is a major worldwide health problem, and chronically infected individuals are at high risk for developing cirrhosis and hepatocellular carcinoma. 1,2 Despite the availability of an HBV vaccine, there are still more than 350 million chronically infected people worldwide, and the few antiviral treatments currently available have a limited rate of efficacy. The narrow host range of HBV and the lack of both in vitro systems and of convenient animal models have greatly hampered our understanding of the complete virus life cycle, as well as the development of more effective antiviral drugs aimed at eradicating the virus from chronic carriers. 3 Chimpanzees are the only animal species infectable with HBV, 4,5 but studies with these animals and evaluation of antiviral therapies are severely restricted because of their limited availability and high costs. Animal models based on HBV-related hepadnaviruses, such as woodchuck and Pekin duck hepatitis B viruses, are often used for assessment of antiviral drugs 6-8 and have provided important information about factors involved in establishment of virus infection, viral persistence, and hepatocarcinogenesis. 9-14 However, woodchucks are relatively large animals of outbred origins that are difficult to handle in many laboratories, and chronic hepadnavirus infection in birds does not lead to cancer. The development of HBV-expressing transgenic mice has also provided important insights regarding viral pathobiology and the role of HBV gene products in hepatocellular injury. 12,[15][16][17][18][19] Although infectious virus can be produced in transgenic mice, their hepatocytes are not permissive for infection. Therefore, the still-unknown early step...

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“…Studies in both animals and humans have proven that the G145R mutant can infect the host without the help of a wild type virus (83,84). This suggests that the native conformation of the second ''a'' loop is not essential, at least for the entrance of the virus into the cell.…”

Section: Transmissionmentioning

confidence: 99%

HBsAg Variants: Common Escape Issues

Dindoost¹,

Jazayeri

Karimzadeh

et al. 2012

Jundishapur J Microbiol

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Licensed recombinant hepatitis B vaccines protect chimpanzees against infection with the prototype surface gene mutant of hepatitis B virus

Cited by 107 publications

References 84 publications

Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus

HBsAg Variants: Common Escape Issues

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