Application of a Newly Developed High-Sensitivity HBsAg Chemiluminescent Enzyme Immunoassay for Hepatitis B Patients with HBsAg Seroclearance

We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring.     

Specific mutations in enhancer II/core promoter of hepatitis B virus subgenotypes C1/C2 increase the risk of hepatocellular carcinoma

BACKGROUND/AIMS: Hepatitis B virus genotype C (HBV/C) has been classified into two geographically distinct subgenotypes; HBV/C1/Cs (Southeast Asia) and HBV/C2/Ce (East Asia). METHODS: Viral differences in enhancer II/core promoter and precore regions between the subgenotypes and their association with hepatocellular carcinoma (HCC) were assessed in a matched cross-sectional control study of 118 carriers (from Hong Kong) with HBV/C1/Cs (48.0 years, 81% male, 40% HBeAg+, 44% HCC) and 210 HBV/C2/Ce (172 from Japan, 38 from Hong Kong) (50.2 years, 78% male, 30% HBeAg+, 46% HCC). RESULTS: Univariate analyses showed that mutation V1753 was predictive for HCC among HBeAg-positive-C1/Cs-carriers (P=0.0055), and T1653 among HBeAg-positive-C2/Ce-carriers (P=0.018), and T1653 or V1753 or T1762/A1764 among HBeAg-negative-C2/Ce-carriers (P<0.05). In the multivariate analysis on all HBV/C subjects, independent predictive factors for HCC were subgenotype C2/Ce (odds ratio, 4.21; 95% confidence interval, 1.07-16.23), T1653 (3.64; 1.93-6.86), V1753 (3.07; 1.66-5.65) and T1762/A1764 (2.58; 1.21-5.49) mutations, age (50 years), gender (male) and HBeAg (positive). CONCLUSIONS: Our data indicate that T1653 and/or V1753 mutations in addition to T1762/A1764 are differently associated with HCC in context of HBeAg status among HBV/C1/Cs and C2/Ce-carriers. HBV/C subgenotypes have specific mutation patterns, which is probably responsible for increased carcinogenesis of HBV/C2/Ce.     

Molecular epidemiology of hepatitis B virus in the United Republic of Tanzania

In the United Republic of Tanzania, 457 voluntary blood donors were enrolled in hepatitis B virus (HBV) serological screening; 4.8% (22/457) carried HBsAg, 13.6% (3/22) of whom were HBeAg-positive. The mean age among HBeAg-negative carriers was 31 years. HBV DNA was detectable in 81.8% (18/22), the mean level was 3.67 (+/-1.77) log copies/ml. Genotype A was determined in 90.9% (20/22) and 18/20 were classified into subgenotype Aa (Asia/Africa). The basal core promoter, precore and partial core nucleotide sequences were analyzed in the 18 strains; T1809/T1812 ("Kozak" sequence) and A/T1888 (encapsidation signal) variants were identified in 100% and 78%, respectively. The complete genome sequencing for one of the Tanzanian strains revealed no recombination. In conclusion, HBV seroprevalence is high among general population in Tanzania, and the HBV/Aa-infection is predominant. The indicated tendency to early HBeAg seroconversion and declining of the viral load should be confirmed further in case-control studies.     

Case–control study for the identification of virological factors associated with fulminant hepatitis B

Background:  Host and viral factors can promote the development of fulminant hepatitis B (FHB), but there have been no case–control studies for figuring out virological parameters that can distinguish FHB.
Methods:  In a case–control study, virological factors associated with the development of FHB were sought in 50 patients with FH developed by transient hepatitis B virus (HBV) infection (FH-T) and 50 with acute self-limited hepatitis B (AHB) who were matched for sex and age. In addition, 12 patients with FH developed by acute exacerbation (AE) of asymptomatic HBV carrier (ASC) (FH-C) were also compared with 12 patients without FH by AE of chronic hepatitis B (AE-C).
Results:  Higher HBV DNA levels, subgenotype B1/Bj, A1762T/G1764A, G1896A, G1899A and A2339G mutation were significantly more frequent ( P  < 0.05), while hepatitis B e-antigen was less frequent in the FH-T patients than AHB. In multivariate analysis, G1896A mutation (odds ratio [OR], 13.53; 95% confidence interval [CI], 2.75–66.64), serum HBV DNA more than 5.23 log copies/mL (OR, 5.14; 95% CI, 1.10–24.15) and total bilirubin more than 10.35 mg/mL (OR, 7.81; 95% CI, 1.77–34.51) were independently associated with a fulminant outcome by transient HBV infection. On the other hand, in comparison with the patients between FH-C and AE-C groups, there was no significant difference of virological factors associated with the development of FHB.
Conclusion:  A number of virological factors have been defined that may distinguish FH-T from AHB in a case–control study. The pathogenic mechanism of FHB between transient HBV infection and AE of ASC would be different.     

Induction of potent cellular immune response in mice by hepatitis C virus NS3 protein with double-stranded RNA

Double-stranded RNA is produced during virus replication and, together with the viral antigen, is responsible for inducing host antivirus immunity. The hepatitis C virus (HCV) non-structural protein-3 (NS3) has been implicated in the immune evasion of HCV, and is one of the prime targets for inducing immunity against HCV infection. Mice were immunized with recombinant NS3 protein (rNS3) and poly (I:C) emulsified in Montanide ISA 720 (M720). Cytokine production was assayed by enzyme-linked immunospot assay, and CD4(+) IFN-gamma(+) T helper (Th) cells or CD8(+) IFN-gamma(+) cytotoxic T lymphocytes were detected by flow cytometry. Anti-NS3 titre and immunoglobulin G2a (IgG2a) and IgG1 levels were monitored by enzyme-linked immunosorbent assay. Administration of rNS3 formulated in poly (I:C) and M720 induced anti-NS3 titres with a predominantly IgG2a isotype comparable to those induced by rNS3 in CpG-ODN and M720. The cytokine profiles showed that this formulation induced a Th1-biased immune response with several-fold more interferon-gamma (IFN-gamma)-producing cells than interleukin-4-producing cells. In contrast, rNS3 in M720 induced a Th2-biased immune response. The frequency of IFN-gamma-producing CD4(+) and CD8(+) cells induced by rNS3 in poly (I:C) and M720 was significantly higher than that induced by rNS3, rNS3 in M720, or rNS3 in poly (I:C), and was comparable to that induced by rNS3 in CpG-ODN with M720. The antigen-specific CD8(+) T-cell immune response persisted for up to 7 months after immunization. In conclusion, poly (I:C) with rNS3 in M720 can elicit a strong and persistent Th1-biased immune response and a cytotoxic T-lymphocyte response through cross-priming in mice. This study highlighted a promising formulation for inducing an efficient cellular immune response against HCV that has potential for HCV vaccine development.     

Differences of hepatocellular carcinoma patients with hepatitis B virus genotypes of Ba, Bj or C in Japan

Hepatitis B virus (HBV) genotypes B (HBV/B) and C (HBV/C) are prevalent in Asia. Recently HBV/B has been classified into two subtypes, HBV/Ba which is ubiquitously found in Asia, and HBV/Bj which is specific in Japan. In addition, the frequency of positive HBeAg has been reported to be higher in patients with HBV/Ba than those with HBV/Bj. However, little is known about the differences between patients with various genotypes who developed hepatocellular carcinoma (HCC). In 296 serum samples of HCC patients collected from all over Japan, HBV genotypes were determined with the restriction fragment length polymorphism. HBV/A was detected in 1.0%, HBV/Ba in 4.4%, HBV/Bj in 7.4%, and HBV/C in 86.5%. In the Tohoku district and Okinawa, HBV/Ba, HBV/Bj and HBV/C were found in 6.7, 40.0 and 48.9%, compared to 4.0, 1.6 and 93.2% in the other districts in Japan. HBV/Bj patients were more frequently found in the group older than 65 years while HBV/Ba patients were found in all age groups. The frequency of positive HBeAg in HBV/Bj patients was significantly low compared to that in the other patients. More than 60% of the patients with HCC had cirrhosis as the underlying liver diseases. However, in HBV/Ba patients aged 50 years or younger, 80% of them had chronic hepatitis, while 87.5% of those aged older than 50 years had cirrhosis. These data suggest that great differences exist among patients with HCC infected with different genotypes.