Do the circulating Pre-S/S quasispecies influence hepatitis B virus surface antigen levels in the HBeAg negative phase of HBV infection?

BACKGROUND: Virus, host factors and their interplay influence Hepatitis B surface Antigen serum levels during Hepatitis B Virus (HBV) infection course and treatment. AIM: To study the Pre-S/S circulating quasispecies in a cohort of untreated, HBeAg negative, genotype-D, HBsAg carriers. METHODS: We studied 260 carriers: 71 with HBeAg negative infection (ENI; HBV-DNA ≤2000 IU/mL); 42 Grey Zone (GZ; HBV-DNA ≤20 000 IU/mL); 82 chronic hepatitis (CH) and 65 cirrhosis (CI) (HBV-DNA > 20 000 IU/mL). Population sequencing was applied to identify Pre-S/S gene mutations responsible for any amino acid substitution or potential biological/antigenic implications (M-muts) on HBsAg. RESULTS: HBsAg serum levels were lower in ENI + GZ than in CH + CI (2.61 [-1.10/4.06] vs 3.62 [2.41/4.92] log10 IU/mL, P < 0.001) and in CI than CH (3.48 [2.41/4.38] vs 3.66 [2.57/4.92] log10 IU/mL, P < 0.001). M-muts were found in 73 (28.1%) cases: 5 (7.0%) ENI, 3 (7.1%) GZ, 26 (31.7%) CH, 39 (60.0%) CI (P < 0.001) and mostly in Pre-S2 (17.6%) than Pre-S1 (5.8%) and Small-S (10.8%; P < 0.001). Overall HBsAg serum levels were higher in carriers with M-muts (3.56 [0.95/4.38] vs 3.17 [-1.10/4.92] log10 IU/mL, P < 0.001), but comparable in carriers with or without M-mut when considering separately ENI + GZ (2.84 [0.95/3.89] vs 2.61 [-1.10/4.06] log10 IU/mL, P = 0.330] and CH + CI (3.57 [2.67/4.38] vs 3.63 [2.41/4.92] log10 IU/mL, P = 0.37). Infection phase (ß: 0.422, P < 0.001), age (ß: -0.260, P < 0.001), ALT (ß: -0.103, P = 0.045), liver stiffness (ß: -0.118, P = 0.039) and HBV-DNA (ß: 0.384, P < 0.001), but not M-mut were independently associated with HBsAg serum levels. CONCLUSIONS: In HBeAg negative, genotype-D, carriers Pre-S/S heterogeneity increases with severity of liver disease, but does not influence HBsAg serum levels, that in low viraemic carriers are associated with an effective control of HBV.

HBV pre-core mutant in genotype-D infected children is selected during HBeAg/anti-HBe seroconversion and leads to HBeAg negative chronic hepatitis B in adulthood.

Selection of HBeAg defective HBV mutants (mt) during childhood might influence infection outcome in adults. Aim of this study was to correlate the dynamics of pre-core HBV mutant (pre-C mt) selection with virological/clinical outcomes in children followed-up until adulthood. Eighty subjects (50-M/30-F), 70 HBeAg-positive (87.5%), and 10 (12.5%) HBeAg-negative/anti-HBe-positive at the admission, mostly genotype D infected (91.2%), with median age of 6.5 (range: 0.2-17) years, were followed-up for 14.3 years (range: 1.1-24.5); 46 (57.5%) received IFN treatment. HBV-DNA and q-HBsAg were tested by commercial assays, Pre-Core 1896 mt by direct-sequence, oligo-hybridization-assay, and allele-specific-PCR (sensitivity: 30%, 10%, and 0.1% of total viremia). HBeAg/anti-HBe seroconversion (SC) occurred in 55/70 (78.6%) children. After SC, 8 (14.6%) developed HBeAg-negative chronic hepatitis (CHB), 41 (74.5%) remain with HBeAg-negative chronic infection, and 6 (10.9%) lost HBsAg. Baseline HBV-DNA and HBsAg were lower in SC than in no-SC children (median: 7.35 vs 8.95 Log IU/mL, P = 0.005, and 4.72 vs 5.04 Log IU/mL, P = 0.015). The prevalence of pre-C mt increased rapidly (10-40%) around SC. Eventually, pre-C mt was detected in 100% of CHB, in 33% of chronic infections without disease, and in 16% of subjects who cleared HBsAg (P < 0.001). HBV-DNA levels remained slightly higher in carriers of HBeAg negative infection with dominant/mixed pre-C mt populations, than in those with dominant pre-C wt (mean Log IU/mL: 3.83 and 3.42 vs 2.67, P = 0.007). In conclusion, pre-C-mt is selected during HBeAg/anti-HBe SC in children with poor control of HBV replication, leading to HBeAg-negative chronic-active-hepatitis during adulthood.

Long-term outcome of inactive and active, low viraemic HBeAg-negative-hepatitis B virus infection: Benign course towards HBsAg clearance.

BACKGROUND & AIMS: The difference between the long-term outcome of low-viraemic (HBV-DNA≤20 000-IU/mL, LV-AC) and inactive HBsAg carriers (HBV-DNA≤2000-IU/mL, IC) remains to be defined. We studied prospectively 153 HBeAg-negative HBsAg-carriers with baseline HBV-DNA≤20 000-IU/mL and normal transaminases. METHODS: IC, LV-AC or chronic hepatitis B (CHB) (HBV-DNA persistently ≤2000-IU/mL, ≤20 000-IU/mL or >20 000-IU/mL respectively) were diagnosed after 1-year, 3-monthly monitoring. Thereafter IC and LV-AC were followed-up for additional 57.2 (8.5-158.3) months. HBV-DNA, HBsAg, HBV"core-related"Antigen (HBcrAg) and total-anti-HBc were quantified at baseline. RESULTS: After the 1st year diagnostic follow-up CHB [higher HBV-DNA (P=.005), total-anti-HBc (P=.012), ALT (P=.007) and liver-stiffness (P=.021)] was identified in 20 (13.1%) carriers; baseline HBsAg≤1000IU/HBV-DNA≤2000IU/mL excluded the presence of CHB (NPV-100%). Thereafter, during the long-term follow-up none of 87 IC reactivated, 19 (21.8%) cleared HBsAg [older-age (P=.004), lower HBsAg (P<.001), higher yearly HBsAg decline (P<.001)]. Twenty-five of 46 (54.3%) LV-AC remained stable, 20 (43.5%) became IC and 1 (2.2%) developed CHB. The best single-point CHB and IC diagnostic-accuracies were total-anti-HBc (84.2%, NPV-98.2%) and HBV-DNA/total-anti-HBc/HBcrAg combination (89.5%, 93%-sensitivity, 84.8%-specificity) respectively. CONCLUSIONS: Viraemia persistently ≤20 000-IU/mL predicts a benign clinical outcome: it was associated with transition to IC in 43% of LV-AC and to Occult HBV Infection in 20% of IC within 5-years. Nevertheless, 13.1% of individuals with low viraemia at presentation develops CHB within 1 year: 1-year HBV-DNA monitoring resulted the most accurate diagnostic approach that can be limited to at least a half of cases by the single point HBV-DNA/HBsAg quantification. The IC-diagnostic-accuracy combining HBV-DNA/total-anti-HBc/HBcrAg needs to be confirmed in further studies.

Total Hepatitis B Core Antigen Antibody, a Quantitative Non-Invasive Marker of Hepatitis B Virus Induced Liver Disease.

Non invasive immunologic markers of virus-induced liver disease are unmet needs. We tested the clinical significance of quantitative total and IgM-anti-HBc in well characterized chronic-HBsAg-carriers. Sera (212) were obtained from 111 HBsAg-carriers followed-up for 52 months (28-216) during different phases of chronic-HBV-genotype-D-infection: 10 HBeAg-positive, 25 inactive-carriers (HBV-DNA≤2000IU/ml, ALT<30U/L), 66 HBeAg-negative-CHB-patients and 10 with HDV-super-infection. In 35 patients treated with Peg-IFN±nucleos(t)ide-analogues (NUCs) sera were obtained at baseline, end-of-therapy and week-24-off-therapy and in 22 treated with NUCs (for 60 months, 42-134m) at baseline and end-of-follow-up. HBsAg and IgM-anti-HBc were measured by Architect-assays (Abbott, USA); total-anti-HBc by double-antigen-sandwich-immune-assay (Wantai, China); HBV-DNA by COBAS-TaqMan (Roche, Germany). Total-anti-HBc were detectable in all sera with lower levels in HBsAg-carriers without CHB (immune-tolerant, inactive and HDV-superinfected, median 3.26, range 2.26-4.49 Log10 IU/ml) versus untreated-CHB (median 4.68, range 2.76-5.54 Log10 IU/ml), p<0.0001. IgM-anti-HBc positive using the chronic-hepatitis-cut-off" (0.130-S/CO) were positive in 102 of 212 sera (48.1%). Overall total-anti-HBc and IgM-anti-HBc correlated significantly (p<0.001, r=0.417). Total-anti-HBc declined significantly in CHB patients with response to Peg-IFN (p<0.001) and in NUC-treated patients (p<0.001); the lowest levels (median 2.68, range 2.12-3.08 Log10 IU/ml) were found in long-term responders who cleared HBsAg subsequently. During spontaneous and therapy-induced fluctuations of CHB (remissions and reactivations) total- and IgM-anti-HBc correlated with ALT (p<0.001, r=0.351 and p=0.008, r=0.185 respectively). Total-anti-HBc qualifies as a useful marker of HBV-induced-liver-disease that might help to discriminate major phases of chronic HBV infection and to predict sustained response to antivirals.

Immunotherapy of HCC metastases with autologous T cell receptor redirected T cells, targeting HBsAg in a liver transplant patient.

HBV-DNA integration frequently occurs in HBV-related hepatocellular carcinoma (HCC), but whether HBV antigens are expressed in HCC cells and can be targeted by immune therapeutic strategies remains controversial. Here, we first characterized HBV antigen expression in HCC metastases, occurring in a patient who had undergone liver transplantation for HBV-related HCC. We then deployed for the first time in HCC autologous T cells, genetically modified to express an HBsAg specific T cell receptor, as therapy against chemoresistant extrahepatic metastases. We confirmed that HBV antigens were expressed in HCC metastases (but not in the donor liver) and demonstrated that tumour cells were recognized in vivo by lymphocytes, engineered to express an HBV-specific T cell receptor (TCR). Gene-modified T cells survived, expanded and mediated a reduction in HBsAg levels without exacerbation of liver inflammation or other toxicity. Whilst clinical efficacy was not established in this subject with end-stage metastatic disease, we confirm the feasibility of providing autologous TCR-redirected therapy against HCC and advocate this strategy as a novel therapeutic opportunity in hepatitis B-associated malignancies.

A serum microRNA signature is associated with the immune control of chronic hepatitis B virus infection.

BACKGROUND AND AIMS: The virus/host interplay mediates liver pathology in chronic HBV infection. MiRNAs play a pivotal role in virus/host interactions and are detected in both serum and HBsAg-particles, but studies of their dynamics during chronic infection and antiviral therapy are missing. We studied serum miRNAs during different phases of chronic HBV infection and antiviral treatment. METHODS: MiRNAs were profiled by miRCURY-LNA-Universal-RT-miRNA-PCR (Exiqon-A/S) and qPCR-panels-I/II-739-miRNA-assays and single-RT-q-PCRs. Two cohorts of well-characterized HBsAg-carriers were studied (median follow-up 34-52 months): a) training-panel (141 sera) and HBsAg-particles (32 samples) from 61 HBsAg-carriers and b) validation-panel (136 sera) from 84 carriers. RESULTS: Thirty-one miRNAs were differentially expressed in inactive-carriers (IC) and chronic-hepatitis-B (CHB) with the largest difference for miR-122-5p, miR-99a-5p and miR-192-5p (liver-specific-miRNAs), over-expressed in both sera and HBsAg-particles of CHB (ANOVA/U-test p-values: <0.000001/0.000001; <0.000001/0.000003; <0.000001/0.000005, respectively) and significantly down-regulated during- and after-treatment in sustained-virological-responders (SVR). MiRNA-profiles of IC and SVR clustered in the heatmap. Liver-miRNAs were combined with miR-335, miR-126 and miR-320a (internal controls) to build a MiR-B-Index with 100% sensitivity, 83.3% and 92.5% specificity (-1.7 cut-off) in both training and validation cohorts to identify IC. MiR-B-Index (-5.72, -20.43/14.38) correlated with ALT (49, 10/2056 U/l, ρ = -0.497, p<0.001), HBV-DNA (4.58, undetectable/>8.3 Log10 IU/mL, ρ = -0.732, p<0.001) and HBsAg (3.40, 0.11/5.49 Log10 IU/mL, ρ = -0.883, p<0.001). At multivariate analysis HBV-DNA (p = 0.002), HBsAg (p<0.001) and infection-phase (p<0.001), but not ALT (p = 0.360) correlated with MiR-B-Index. In SVR to Peg-IFN/NUCs MiR-B-Index improved during-therapy and post-treatment reaching IC-like values (5.32, -1.65/10.91 vs 6.68, 0.54/9.53, p = 0.324) beckoning sustained HBV-immune-control earlier than HBsAg-decline. CONCLUSIONS: Serum miRNA profile change dynamically during the different phases of chronic HBV infection. We identified a miRNA signature associated with both natural-occurring and therapy-induced immune control of HBV infection. The MiR-B-Index might be a useful biomarker for the early identification of the sustained switch from CHB to inactive HBV-infection in patients treated with antivirals.

Lamivudine-resistant HBV strain rtM204V/I in acute hepatitis B.

AIMS: To detect HBV rtM204V/I lamivudine-resistant strains in serum of patients with acute hepatitis B and to assess their biological and clinical significance. METHODS: Eighty HBV DNA-positive patients with symptomatic acute hepatitis B observed from 1999 to 2010 were enrolled. A plasma sample obtained at the first observation was tested for HBV mutants in the polymerase region by direct sequencing; the antiviral drug-resistant rtM204V/I mutations, the most frequent HBV mutants in Italy, were also sought by the more sensitive allele-specific polymerase chain reaction (PCR). RESULTS: No HBV mutation associated with resistance to nucleos(t)ide analogues was identified by direct sequencing, whereas allele-specific PCR identified HBV strains carrying the substitution rtM204V/I in 11 (13.7%) patients. Compared with those with the HBV wild strain, patients with rtM204V/I more frequently showed severe acute hepatitis B (36.4% vs 8.7%; p < 0.05) and lower values of serum HBV DNA (1.77 × 10(6) ± 4.76 × 10(6) vs. 1.68 × 10(8) ± 5.46 × 10(8)). In addition, a multivariate analysis identified the presence of a pre-existing HCV chronic infection as independently associated with severe acute hepatitis B (p < 0.05). CONCLUSIONS: HBV rtM204V/I lamivudine-resistant strains were detected in serum of 11 (13.7%) patients with acute hepatitis B by allele-specific polymerase chain reaction. The frequent association of rtM204V/I with a more severe acute hepatitis B and with a lower viral load may suggest that greater and/or more prolonged immune pressure might have induced their selection.

Bioinformatics methods for the analysis of hepatitis viruses.

HBV and HCV are the only hepatotropic viruses capable of establishing chronic infections. More than 500 million people worldwide are estimated to have chronic infections with HBV and/or HCV, and they have an increased risk of developing liver complications, such as cirrhosis or hepatocellular carcinoma. During the past decade, several antiviral agents including immune-modulatory drugs and nucleoside/nucleotide analogues have been approved for the treatment of HBV and HCV infections. In recent years, the focus has been on the development of new and better therapeutic agents for management of chronic HCV infections. Bioinformatics has only been applied recently to the field of viral hepatitis research. In addition to the wide range of general tools freely available for identification of open reading frames, gene prediction, homology searching, sequence alignment, and motif and epitope recognition, several public database systems designed specifically for HBV and HCV research have now been developed. The focus of these databases ranged from being viral sequence repositories for the provision of bioinformatics tools for viral genome analysis, as well as HBV or HCV drug resistance prediction. This review provides an overview of these public databases, which have integrated bioinformatics tools for HBV and HCV research. Properly managed and developed, these databases have the potential to have a broad effect on hepatitis research and treatment strategies. However, the effect will depend on the comprehensive collection of not only molecular sequence data, but also anonymous patient clinical and treatment data.

Optimization of in vitro HBV replication and HBsAg production in HuH7 cell line.

The Gunther's vector-free method (GM), using PCR-amplified full length HBV-DNA (fl-HBV-DNA), is currently the best in vitro HBV replication system despite the low intracellular HBV-DNA production. The replication efficiency and HBsAg secretion of 12 isolates from HBsAg/HBeAg positive sera by GM, Monomer-Linear-Sticky-Ends-DNA (MLSE) and Monomer-Circular-Closed (MCC) were compared in HuH7 cells. Eight of twelve genomes (67%) were replication competent by GM; however direct sequencing (DS) showed that more than 80% of input DNA was undigested in spite of SapI treatment. Replication Intermediates (RI) were detected earlier (24 vs. 48h) and in higher amounts (2.51±0.32 and 6.43±0.43 fold) by MCC than GM or MLSE. By MCC 10 of 12 genomes (83%) were replication competent and 7 produced high RI levels. RI and HBsAg kinetics correlated positively in MCC (R=0.696, p=0.017 overall; R=0.928, p=0.008), but not in GM (R=-0.437, p=0.179 overall; R=-0.395, p=0.439) in genotype D isolates. In conclusion, HBV-DNA circularization prior transfection improves in vitro viral replication and replication competent HBsAg production, mimicking better the in vivo conditions.

Hepatitis B surface antigen serum levels help to distinguish active from inactive hepatitis B virus genotype D carriers.

BACKGROUND & AIMS: The accurate identification of inactive (serum HBV-DNA persistently or=20,000 IU/mL (AC2): 883 (0.5-7838) vs 4233 (164-82,480) IU/mL, P=.002. HBV infection was less productive in IC and AC1 than AC2 (log10 HBV-DNA/HBsAgsl ratios 0.25 and 0.49 vs 2.06, respectively, P<.001) and in chronic hepatitis than cirrhosis (1.97 vs 2.34, respectively; P=.023). The combined single point quantification of HBsAg (<1000 IU/mL) and HBV-DNA (

Hepatitis B virus surface antigen levels: a guide to sustained response to peginterferon alfa-2a in HBeAg-negative chronic hepatitis B.

UNLABELLED: We investigated the relationship between hepatitis B virus surface antigen (HBsAg) serum level decline and posttreatment response in patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B from a large multinational study of pegylated interferon alfa-2a (peginterferon alfa-2a), with or without lamivudine, versus lamivudine alone. Serum HBsAg was quantified using the Architect assay (Abbott Diagnostics) at pretreatment, end of treatment (week 48), and 6 months after the end of treatment (week 72) in sera from 386 of the 537 patients who participated in the multinational study (peginterferon alfa-2a, 127; peginterferon alfa-2a plus lamivudine, 137; lamivudine monotherapy, 122). Pretreatment HBsAg levels varied according to genotype, with the highest levels present in patients infected with genotypes A (median, 4.11 log(10) IU/mL) and D (median, 3.85 log(10) IU/mL). Significant on-treatment decline in HBsAg was observed during treatment with peginterferon alfa-2a (alone or combined with lamivudine; mean decline at week 48, -0.71 and -0.67 log(10) IU/mL, respectively, P < 0.001), but not during treatment with lamivudine alone (-0.02 log(10) IU/mL). Significantly more patients treated with peginterferon alfa-2a (21%) or peginterferon alfa-2a plus lamivudine (17%) achieved HBsAg levels <100 IU/mL at the end of treatment compared with lamivudine (1%) (both P < 0.001 versus lamivudine). End-of-treatment HBsAg level correlated strongly with HBV DNA suppression to 1 log(10) IU/mL were significantly associated with sustained HBsAg clearance 3 years after treatment (both P < 0.0001). CONCLUSION: On-treatment quantification of HBsAg in patients with HBeAg-negative chronic hepatitis B treated with peginterferon alfa-2a may help identify those likely to be cured by this therapy and optimize treatment strategies.

Viral load 1 week after liver transplantation, donor age and rejections correlate with the outcome of recurrent hepatitis C.

BACKGROUND: Early identification of patients at a higher risk of rapidly progressive recurrent hepatitis post liver transplantation (LT) could help to tailor antiviral therapy. METHODS: We studied the correlation between early post-LT viral load and the histological and clinical outcomes of 49 consecutive patients (34 males, median age 55 years) in whom viraemia was monitored at days 0, 1, 7, 30, 180 and 365 after LT. RESULTS: Hepatitis C recurred at histology in 38 of 42 (90.5%) patients. Early viral load after LT was higher in patients with rapidly progressive hepatitis C recurrence (day 7 median HCV-RNA levels: 5.84 vs 4.93 Log(10) IU/ml, P=0.003). Day 7 HCV-RNA levels >/=2.5 x 10(5) IU/ml, donor age >60 years and rejection episodes were independently associated with progression to cirrhosis within one year post-LT [P=0.018, odds ratio (OR) 27.59; P=0.043, OR 13.85 and P=0.048, OR 9.95, respectively]. Day 7 viraemia and rejection episodes were independently associated with 5-years survival. Day 7 viraemia, in combination with acute hepatitis and/or donor age, showed 80% sensitivity, 94% specificity and 90.5% diagnostic accuracy to identify severe recurrence. CONCLUSIONS: Early post-LT HCV-RNA correlates with the severity of hepatitis C recurrence and in combination with donor age (>60 years) and rejections, identifies patients with a high risk of severe recurrence and candidates of cost-effective pre-emptive antiviral therapy.