Ccc DNA Research Articles

NTCP-Reconstituted In Vitro HBV Infection System.

Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for hepatitis B virus (HBV). Expressing human NTCP in human hepatoma HepG2 cells (HepG2-NTCP) renders these cells susceptible for HBV infection. The HepG2-NTCP stably transfected cell line provides a much-needed and easily accessible platform for studying the virus. HepG2-NTCP cells could also be used to identify chemicals targeting key steps of the virus life cycle including HBV covalent closed circular (ccc) DNA, and enable the development of novel antivirals against the infection.Many factors may contribute to the efficiency of HBV infection on HepG2-NTCP cells, with clonal differences among cell line isolates, the source of viral inoculum, and infection medium among the most critical ones. Here, we provide detailed protocols for efficient HBV infection of HepG2-NTCP cells in culture; generation and selection of single cell clones of HepG2-NTCP; production of infectious HBV virion stock through DNA transfection of recombinant plasmid that enables studying primary clinical HBV isolates; and assessing the infection with immunostaining of HBV antigens and Southern blot analysis of HBV cccDNA.

Clinical implication of occult hepatitis B infection

Occult hepatitis B infection (OBI) can be found in three different clinical groups of patients according to the documentation of the history of hepatitis B virus (HBV) exposure. They are patients with definite history of apparently self-limiting acute hepatitis B infection, patients with known chronic hepatitis B (CHB) infection with loss of hepatitis B surface antigen (HBsAg) (HBsAg seroclearance) and patients without prior history of hepatitis B infection. The second group likely contributes the largest group of patient population in OBI. These patients have same rate of development of end-staged liver disease including cirrhosis-related complications and hepatocellular carcinoma (HCC) if HBsAg seroclearance occurs when there is pre-existing cirrhosis or at the age older than 50 years. Nevertheless, majority of the patients with OBI usually have normal liver biochemistry and histology. The intrahepatic HBV DNA is detectable in almost all the patients whereas the covalently closed circular (ccc)DNA and pre-genomic RNA levels are usually very low. With regard to the clinical implications, HBV transmission from the blood products of occult hepatitis B donors is possible as demonstrated in animal and human experiments, although the actual HBV transmission rate in human is quite low especially in anti-HBs positive recipients. HBV can also be transmitted to the recipients through organ transplantation especially liver transplantation from occult hepatitis B donors. OBI may be an accelerating risk factor for development of HCC in patients with chronic hepatitis C infection and patients with alcoholic liver disease. Importantly, OBI may also contribute to a great extent in patients with cryptogenic cause of HCC. In another scenario, patients with OBI may have severe hepatitis B reactivation with a surge of viral replication and hence clinical hepatitis and mortality. This phenomenon is more commonly seen in patients receiving immunosuppressive therapy especially anti-CD monoclonal antibody. Patients receiving haematopoetic stem cell transplantation also carry a high risk of hepatitis B reactivation. Prompt antiviral therapy at the time of detectable HBV DNA upon interval monitoring is very effective in controlling hepatitis B viral replication and therefore is associated with a hepatitis-free recovery.

Ethics and hepatitis B cure research

Recent scientific advances, including the development of curative therapies for HCV and the establishment of global cure initiatives for HIV, have led to international calls seeking a cure for chronic...

HBV COVALENTLY CLOSED CIRCULAR DNA AS A MARKER OF PREVALENCE OF OCCULT HEPATITIS В IN PATIENTS WITH HBV, HDV AND HCV INFECTION IN UZBEKISTAN

Aim. Evaluate significance of covalently closed circular DNA of hepatitis В virus as a marker for detection of occult viral hepatitis В in Uzbekistan population with hepatitis of various genesis. Materials and methods. Blood plasma and liver biopsy from 39 patients with different severity levels of liver fibrosis and cirrhosis served as study material. HBV covalently closed circular DNA detection was carried out according to Pollicino T. et al. (2004). Results. Covalently closed circular DNA of hepatitis В virus was detected in 82% of samples, including in 54.5% of patients with chronic viral hepatitis C (CVHC) and in 100% of patients with hepatitis of unknown etiology. Quantitative evaluation of content of covalently closed circular DNA of hepatitis В virus in liver tissue in patients with CVHB has shown an average of 2.5 copies of HBV genome as ccc DNA per cell, in patients with CVHB + D an average of 0.7 copies/cell, in patients with co-infection by HCV and HBV - 0.5 copies/cell, in patients with CVHC an average of 0.12 copies/cell, and in patients with cryptogenic hepatitis - 0.2 copies/cell. Conclusion. Detection of HBV DNA is a complex problem for effective laboratory diagnostics of hepatitis. Detection of H BV ccc DNA as a marker of occult hepatitis В in patients with CVHC and patients with hepatitis of unclear etiology is an important factor for diagnostics, selection of adequate therapy, prognosis of disease outcome and prevention of development of severe liver diseases.

DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.

Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.

Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators

Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is essential to the virus life cycle, its elimination during chronic infection is considered critical to a durable therapy but has not been achieved by current antivirals. Despite being essential, cccDNA has not been the major target of high throughput screening (HTS), largely because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself. In response to this need, we have previously developed a proof-of-concept HepDE19 cell line in which the production of wildtype e antigen (HBeAg) is dependent upon cccDNA. However, the existing assay system is not ideal for HTS because the HBeAg ELISA cross reacts with a viral HBeAg homologue, which is the core antigen (HBcAg) expressed largely in a cccDNA-independent fashion in HepDE19 cells. To further improve the assay specificity, we report herein a “second-generation” cccDNA reporter cell line, termed HepBHAe82. In the similar principle of HepDE19 line, an in-frame HA epitope tag was introduced into the precore domain of HBeAg open reading frame in the transgene of HepBHAe82 cells without disrupting any cis-element critical for HBV replication and HBeAg secretion. A chemiluminescence ELISA assay (CLIA) for the detection of HA-tagged HBeAg with HA antibody serving as capture antibody and HBeAb serving as detection antibody has been developed to eliminate the confounding signal from HBcAg. The miniaturized HepBHAe82 cell based assay system exhibits high level of cccDNA-dependent HA-HBeAg production and high specific readout signals with low background. We have also established a HepHA-HBe4 cell line expressing transgene-dependent HA-HBeAg as a counter screen to identify HBeAg inhibitors. The HepBHAe82 system is amenable to antiviral HTS development, and can be used to identify host factors that regulate cccDNA metabolism and transcription.

Two-way molecular ligation for efficient conversion of monomeric hepatitis B virus DNA constructs into tandem dimers

Replication of the 3.2-kb hepatitis B virus (HBV) genome is driven by the covalently closed circular (ccc) DNA in the nucleus, from which four classes of co-terminal RNAs are transcribed. Genome replication requires just the 3.5-kb pregenomic RNA, which is terminally redundant. Cloning the full-length HBV genome into a vector disrupts its continuity, thus preventing genome replication at the step of pregenomic RNA transcription. This can be overcome by converting the monomeric construct into a tandem dimer, yet the need to ligate two molecules of the HBV genome with vector DNA makes it inefficient and even unsuccessful. To overcome this problem we partially digested the monomeric construct with the unique restriction enzyme used for cloning, and dephosphorylated the linearized monomer before its ligation with another copy of the HBV genome. Alternatively, the monomer was linearized at another unique restriction site inside the HBV genome, followed by its dephosphorylation and ligation with another copy of the HBV genome linearized at the same site. These approaches of two-way molecular ligation greatly improved the efficiency of dimer formation with about 50% of the bacterial colonies screened harboring tandem dimers.

The effect of peginterferon alpha-2a vs. interferon alpha-2a on intrahepatic covalently closed circular DNA in HBeAg-positive chronic hepatitis B patients

The covalently closed circular DNA (cccDNA), as the template of HBV transcription, plays a key role in the virus infection. The present study aimed to compare the effect of pegylated interferon (IFN)-α-2a with that of conventional IFN-α-2a on intrahepatic covalently closed circular (ccc)DNA in patients with chronic hepatitis B. Seventy-six HBeAg-positive chronic hepatitis B patients were randomly divided into two groups (n=38): group A was treated with interferon alpha-2a (IFN-α-2a) and group B was treated with peginterferon alpha-2a (peg IFN-α-2a). The intrahepatic level of cccDNA and its detection rate, levels of hepatitis B virus (HBV) DNA in liver and serum, histologic inflammation and some biochemistry parameters (alanine aminotransferase, aspartate aminotransferase and total bilirubin levels) were measured. The outcome of 48 weeks therapy showed that the mean level of intrahepatic HBV cccDNA level and its detection rate, the levels of HBV DNA and the histology and biochemistry parameters were significantly decreased following therapy in two groups (P<0.05). While, the reductions in the group treated with peg IFN-α-2a were greater (P<0.05). It may be concluded that the ability of the peg IFN-α-2a to clear and suppress cccDNA and HBV DNA was superior compared with that of conventional IFN-α-2a. Furthermore, the effects of peg IFN-α-2a on histology and biochemistry parameters were also more obvious than conventional IFN-α-2a.

Associations between activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like cytidine deaminase expression, hepatitis B virus (HBV) replication and HBV-associated liver disease (Review).

The hepatitis B virus (HBV) infection is a major risk factor in the development of chronic hepatitis (CH) and hepa-tocellular carcinoma (HCC). The activation-induced cytidine deaminase (AID)/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases is significant in innate immunity, as it restricts numerous viruses, including HBV, through hypermutation-dependent and -independent mechanisms. It is important to induce covalently closed circular (ccc)DNA degradation by interferon-α without causing side effects in the infected host cell. Furthermore, organisms possess multiple mechanisms to regulate the expression of AID/APOBECs, control their enzymatic activity and restrict their access to DNA or RNA substrates. Therefore, the AID/APOBECs present promising targets for preventing and treating viral infections. In addition, gene polymorphisms of the AID/APOBEC family may alter host susceptibility to HBV acquisition and CH disease progression. Through G-to-A hypermutation, AID/APOBECs also edit HBV DNA and facilitate the mutation of HBV DNA, which may assist the virus to evolve and potentially escape from the immune responses. The AID/APOBEC family and their associated editing patterns may also exert oncogenic activity. Understanding the effects of cytidine deaminases in CH virus-induced hepatocarcinogenesis may aid with developing efficient prophylactic and therapeutic strategies against HCC.

Detection of total and covalently closed circular HBV DNA in liver transplant recipients due to HBV-associated liver diseases

To investigate the levels of hepatitis B virus (HBV) total DNA and covalently closed circular (ccc) DNA in liver transplant recipients due to HBV-associated liver diseases and detemaine their clinical significance. Sixty patients undergoing liver transplantation (LT) due to HBV-associated liver diseases were enrolled for the study. Levels of HBV total and ccc DNA in plasma, liver and PBMCs were measured by RT-PCR. The ratio of male:female participants was 48:12. The mean age was 52.98+/-9.40 years old, and the median duration post-LT was 72 (25-128) months. 59 of the patients had no detectable HBV DNA in plasma.Four patients had detectable levels of total HBV DNA in PBMCs, but no detectable ccc DNA. Five patients had detectable levels of total HBV DNA in liver, and two of those also had detectable levels of ccc DNA. One patients who had detectable HBV DNA in PBMCs suffered HBV recurrence. The liver transplant recipients with detectable levels of HBV total and ccc DNA in PBMCs and liver should be considered high risk for HBV recurrence following LT.

Retinoic acid-inducible gene 1 and sensing of hepatitis B virus revisited.

Retinoic acid-inducible gene 1 and sensing of hepatitis B virus revisited.

The detection of (total and ccc) HBV DNA in liver transplant recipients with hepatitis B vaccine against HBV reinfection

To investigate the levels of hepatitis B virus total DNA (HBV DNA) and covalently closed circular (ccc) DNA in liver transplant recipients who received hepatitis B vaccination, including responders and non-responders, following liver transplantation due to hepatitis B-related diseases and to investigate the efficacy of hepatitis B immune reconstitution against HBV reinfection. Twenty responders and 34 non-responders were enrolled in the present study. The levels of HBV total DNA and ccc DNA in peripheral blood mononuclear cells (PBMCs) and the liver and plasma were detected by real-time polymerase chain reaction (PCR). Fifty-three blood samples and 38 liver allograft tissues were acquired. For the responders, the mean serum titer for anti-HBs (antibodies against hepatitis B surface antigen) was 289 (46.64–1000) IU/ml. Also for the responders, HBV total DNA was detected in PBMCs for one recipient and in the liver for another recipient, but ccc DNA was not detected in either of those 2 recipients. For the non-responders, HBV total DNA was detected in PBMCS for 2 recipients, neither of whom had ccc DNA. Also for the non-responders, HBV total DNA was detected in the livers of 3 recipients, 2 of whom also had ccc DNA. All responders had discontinued hepatitis B immunoglobulin (HBIG), and 13 responders had discontinued antiviral agents. One responder experienced HBV recurrence during the follow-up period. For the majority of liver transplant recipients, no HBV total DNA or ccc DNA was detected in the blood or liver. The lack of HBV total DNA and ccc DNA both in PBMCs and the liver in liver transplant recipients who received hepatitis B vaccination to prevent HBV reinfection should be a prerequisite for the withdrawal of HBIG and/or antiviral agents.

Spinoculation Enhances HBV Infection in NTCP-Reconstituted Hepatocytes.

Hepatitis B virus (HBV) infection and its sequelae remain a major public health burden, but both HBV basic research and the development of antiviral therapeutics have been hindered by the lack of an efficient in vitro infection system. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the HBV receptor. We herein report that we established a NTCP-complemented HepG2 cell line (HepG2-NTCP12) that supports HBV infection, albeit at a low infectivity level following the reported infection procedures. In our attempts to optimize the infection conditions, we found that the centrifugation of HepG2-NTCP12 cells during HBV inoculation (termed “spinoculation”) significantly enhanced the virus infectivity. Moreover, the infection level gradually increased with accelerated speed of spinoculation up to 1,000g tested. However, the enhancement of HBV infection was not significantly dependent upon the duration of centrifugation. Furthermore, covalently closed circular (ccc) DNA was detected in infected cells under optimized infection condition by conventional Southern blot, suggesting a successful establishment of HBV infection after spinoculation. Finally, the parental HepG2 cells remained uninfected under HBV spinoculation, and HBV entry inhibitors targeting NTCP blocked HBV infection when cells were spinoculated, suggesting the authentic virus entry mechanism is unaltered under centrifugal inoculation. Our data suggest that spinoculation could serve as a standard protocol for enhancing the efficiency of HBV infection in vitro.

Intrahepatic hepatitis B virus covalently closed circular DNA correlation with serum HBV DNA, serum HBsAg, alanine aminotransferase and age

To investigate the correlation between intrahepatic eovalently closed circular (ccc)DNA of hepatitis B virus (HBV) and pathogen-and patient-related parameters. Ultrasound-guided liver biopsies were obtained from 60 patients with chronic HBV infection. Levels of intrahepatic HBV cccDNA and serum HBV DNA were measured by quantitative fluorescence PCR. Level of serum hepatitis B surface antigen (HBsAg) was measured by chemiluminescence immunoassay. Clinical parameters, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (AKP), albumin, globulin (GLO), white blood cell, platelet, prothrombin-international normalized ratio, were measured by standard assay. Demographic information was recorded.The correlation between intrahepatic HBV cccDNA and pathogen-and patient-related parameters was assessed. Intrahepatic HBV cccDNA level was negatively correlated with age, GLO, ALT and grade of necroinflammation. Patients with age of 30 years or more showed significantly higher level of HBV cccDNA level than patients under 30 years-old (7.44±0.58 and 5.66±1.35; t=7.157, P less than 0.001). Intrahepatic HBV cccDNA level was positively correlated with serum HBV DNA level (r=0.916, P less than 0.001) and serum HBsAg level (r=0.727, P less than 0.001). The median ratio of HBV cccDNA to HBV DNA was 1.18, and of HBV cccDNA to HBsAg was 1.67. Intrahepatic HBV cccDNA levels decrease with age, level of ALT, level of GLO and grade of liver necroinflammation, but increase with level of serum HBV DNA and level of serum HBsAg. To a certain extent, serum HBV DNA and serum HBsAg levels may be a sufficient marker of intrahepatic HBV cccDNA levels.

Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease

Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses.

Hepatitis B virus (HBV), the causative agent of chronic hepatitis B and prototypic hepadnavirus, is a small DNA virus that replicates by protein-primed reverse transcription. The product is a 3-kb relaxed circular DNA (RC-DNA) in which one strand is linked to the viral polymerase (P protein) through a tyrosyl-DNA phosphodiester bond. Upon infection, the incoming RC-DNA is converted into covalently closed circular (ccc) DNA, which serves as a viral persistence reservoir that is refractory to current anti-HBV treatments. The mechanism of cccDNA formation is unknown, but the release of P protein is one mandatory step. Structural similarities between RC-DNA and cellular topoisomerase-DNA adducts and their known repair by tyrosyl-DNA-phosphodiesterase (TDP) 1 or TDP2 suggested that HBV may usurp these enzymes for its own purpose. Here we demonstrate that human and chicken TDP2, but only the yeast ortholog of TDP1, can specifically cleave the Tyr-DNA bond in virus-adapted model substrates and release P protein from authentic HBV and duck HBV (DHBV) RC-DNA in vitro, without prior proteolysis of the large P proteins. Consistent with TPD2's having a physiological role in cccDNA formation, RNAi-mediated TDP2 depletion in human cells significantly slowed the conversion of RC-DNA to cccDNA. Ectopic TDP2 expression in the same cells restored faster conversion kinetics. These data strongly suggest that TDP2 is a first, although likely not the only, host DNA-repair factor involved in HBV cccDNA biogenesis. In addition to establishing a functional link between hepadnaviruses and DNA repair, our results open new prospects for directly targeting HBV persistence.

Establishment of an HBV chronic hepatitis B infection mouse model by vivo transduction of HBV cccDNA

To generate a mouse model of chronic hepatitis B (CHB) infection by performing in vivo transduction of hepatitis B virus (HBV) covalently closed circular (ccc)DNA. Nude mice were injected with HBV cccDNA at doses of 1.5, 1.0 or 0.5 mug/ml. A control group was generated by giving equal injection volumes of physiological saline. The serum levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) on post-injection days 1 and 3, weeks 1-6, 8 and 10 were assayed by reflection immunoassay. At post-injection week 10, all animals were sacrificed and liver tissues were collected. Copies of HBV DNA in serum and liver tissue were detected by real-time PCR. HBV antigens in liver tissue were detected of by immunohistochemistry. Pathological analysis of liver tissue carried out with hematoxylin-eosin staining. Linear correlation of data was determined by statistical analysis. HBsAg and HBeAg were detected in sera from all three groups of cccDNA-injected mice staring at post-injection day 1 and lasting through week 10. The levels of HBsAg over the 10-week period showed two patterns of increase-decrease;the lowest level was detected at week 4 and the highest level was detected at week 8. In contrast, the levels of HBeAg over the 10-week period showed three patterns of increase-decrease; the lower levels were detected at weeks 2 and 4 and the higher levels at weeks 3 and 6. HBV DNA copies in liver tissues showed a cccDNA dose-dependent descending trend over the 10-week study period (1.5 mug/ml:1.14E+07 ± 6.51E+06 copies/g, 1.0 mug/ml:9.81E+06 ± 9.32E+06 copies/g, and 0.5 mug/ml:3.72E+06 ± 2.35E+06 copies/g; Pearson's r =0.979). HBV DNA copies in sera showed the pattern of 1.0 mug/ml cccDNA more than 1.5 mug/ml cccDNA more than 0.5 mug/ml cccDNA, and in general were higher than those detected in the liver tissues. Liver tissues from all cccDNA-injected mice showed positive immunohistochemistry staining for both HBsAg and HBeAg. HE staining showed that the liver tissues of all cccDNA-injected mice had severe fatty and vacuolar degeneration and less obvious structure of liver lobules (compared to the liver tissues from control mice). The CHB mouse model successfully established in this study by in vivo transduction of HBV cccDNA may represent a useful tool to study the pathogenic mechanisms and potential antiviral treatments of human CHB.

Quantifiable changes in HBeAg expression predict therapeutic efficacy of peg-interferon alfa-2a in patients with HBeAg-positive chronic hepatitis B

To investigate whether quantifiable changes in serum levels of hepatitis B e antigen (HBeAg) in response to 24 weeks of pegylated-interferon alfa-2a (Peg-IFN-a 2a) treatment are predictive of therapeutic efficacy at 48 weeks of treatment in HBeAg-positive chronic hepatitis B (CHB) patients and to investigate the efficacy of using an individualized antiviral treatment strategy. Ninety-six HBeAg-positive CHB patients with detectable HBeAg at week 24 of Peg-IFN-a 2a treatment were categorized according to the quantitative change in HBeAg (vs. pre-treatment baseline): group A, HBeAg decline more than 2 log; group B, HBeAg decline between 1 - 2 log; group C, HBeAg decline less than 1 log, which was then randomly divided into two sub-groups: C1 and C2. Group A, B, and C1 patients continued the original therapy for an additional 24 weeks, while group C2 patients were supplemented with lamivudine (3TC + Peg-IFN-a 2a) for the additional 24 weeks of treatment. All patients underwent liver biopsy at the end of treatment (week 48), and HBV covalently-closed circular (ccc)DNA was quantified as a measure of therapeutic efficacy. A, B, and C1 between-group multiple comparisons were made by the Nemenyi test; C1 and C2 between-group comparison was made by the Mann-Whitney U test. The significance of between-group differences in decreased HBV cccDNA vs. HBeAg/anti-HBe seroconversion was made by the Chi-squared test. At week 48, the mean decrease of serum HBV cccDNA in each group was: A, 5.8 log10 copy/ml; B, 3.8 log10 copy/ml; C1, 2.8 log10 copy/ml; C2, 5.7 log10 copy/ml. Statistically significant differences were observed for group A vs. B and C1 (P less than 0.01) and C1 vs. C2 (P less than 0.01); however, the difference between group B and C1 did not reach statistical significance (P = 0.19). The mean decrease of HBeAg in each group was: A, 2.7 log10 S/CO; B, 1.9 log10 S/CO; C1, 0.9 log10 S/CO; C2, 1.6 log10 S/CO. Statistically significant differences were observed for group A vs. B and C1 (P less than 0.01) and C1 vs. C2 (P less than 0.01). The rate of patients who achieved undetectable HBV DNA in each group was: A, 87.5%; B, 34.5%; C1, 17.4%; C2, 85.0%. Statistically significant differences were observed for group A vs. B and C1 (P less than 0.01) and C1 vs. C2 (P less than 0.01). The HBeAg seroconversion rates were: A, 75.0%; B, 24.1%; C1, 13.0%; C2, 25.0%. Statistically significant differences were observed only for group A vs. B and C1 (P less than 0.01). Finally, group A achieved greater reduction in levels of cccDNA in liver tissues than B or C1 (P less than 0.01); however, the differences between B and C1 and between C1 and C2 did not reach statistical significance. CHB patients who showed an HBeAg decline of more than 2 log at week 24 of Peg-IFN-a 2a treatment had better treatment outcome at week 48 than those who showed HBeAg decline less than 2 log at week 24. Augmenting the Peg-IFN-a 2a treatment with 3TC can improve the clinical response. A change of quantifiable HBeAg at week 24 of Peg-IFN-a 2a treatment may be a useful predictor of therapeutic efficacy of a 48-week antiviral regimen.

In vivoinfectivity of liver extracts after resolution of hepadnaviral infection following therapy associating DNA vaccine and cytokine genes

DNA-based vaccination appears of promise for chronic hepatitis B immunotherapy, although there is an urgent need to increase its efficacy. In this preclinical study, we evaluated the therapeutic benefit of cytokine (IL-2, IFN-γ) genes co-delivery with DNA vaccine targeting hepadnaviral proteins in the chronic duck hepatitis B virus (DHBV) infection model. Then, we investigated the persistence of replication-competent virus in the livers of apparently resolved animals. DHBV carriers received four injections of plasmids encoding DHBV envelope and core alone or co-delivered with duck IL-2 (DuIL-2) or duck IFN-γ (DuIFN-γ) plasmids. After long-term (8 months) follow-up, viral covalently closed circular (ccc) DNA was analysed in duck necropsy liver samples. Liver homogenates were also tested for in vivo infectivity in neonatal ducklings. Co-delivery of DuIFN-γ resulted in significantly lower mean viremia starting from week 21. Viral cccDNA was undetectable by conventional methods in the livers of 25% and 57% of animals co-immunized with DuIL-2 and DuIFN-γ, respectively. Interestingly, inoculation of liver homogenates from 7 such apparently resolved animals, exhibiting cccDNA undetectable in Southern blotting and DHBV expression undetectable or restricted to few hepatocytes, revealed that three liver homogenates transmitted high-titre viremia (3-5×10(10) vge/mL) to naïve animals. In conclusion, our results indicate that IFN-γ gene co-delivery considerably enhances immunotherapeutic efficacy of DNA vaccine targeting hepadnaviral proteins. Importantly, we also showed that livers exhibiting only minute amounts of hepadnaviral cccDNA could induce extremely high-titre infection, highlighting the caution that should be taken in occult hepatitis B patients to prevent HBV transmission in liver transplantation context.

Elecsys Hepatitis B Surface Antigen Quantitative Assay: Performance Evaluation and Correlation with Hepatitis B Virus DNA during 96 Weeks of Follow-up in Chronic Hepatitis B Patients

BackgroundTreatment for chronic hepatitis B aims to suppress virus replication and virus sequestration in hepatocytes. Covalently closed circular (ccc) DNA is the template for transcription of viral genes and is responsible for viral persistence. However, limited data are available for quantification of hepatitis B surface antigen (HBsAg) in Korea.MethodsWe evaluated the Elecsys HBsAg II quant assay (Roche Diagnostics, USA) for within-run, between-run, and between-day precisions, linearity, carryover, and clinical specificity. In total, 156 serum samples were evaluated for correlation between HBsAg and hepatitis B virus (HBV) DNA. Serial samples were obtained from 10 patients at 0, 12, 24, 48, 72, and 96 weeks during follow-up.ResultsThe assay detected HBsAg in a linear range of 0.5-48,696 IU/mL. Within-run, between-run, and between-day CVs were 2.9-4.1%, 0-1.5%, and 1.5-4.9%, respectively. Cross-reactivity between potentially interfering substances was absent, and the carryover rate was 0.00002%. The correlation of measurements between the Elecsys assay and HBV DNA PCR was weak (r=0.438, P=0.002). For predicting virologic response, cutoff values of 10,275 IU/mL and 3,846 IU/mL at 12 and 24 weeks after treatment initiation showed positive predictive values of 77.1% and 85% and negative predictive values of 84.6% and 50%, respectively.ConclusionsThe Elecsys HBsAg II quant assay showed good performance for precision, linearity, carryover rate, and specificity. HBsAg level at baseline, 12 weeks, and 24 weeks after treatment initiation can predict virologic response, and the assay can be used for HBsAg quantification in clinical practice.