Viral and immune factors associated with successful treatment withdrawal in HBeAg-negative chronic hepatitis B patients.

BACKGROUND & AIMS: Factors associated with a successful outcome upon nucleos(t)ide analogue (NA) treatment withdrawal in HBeAg-negative chronic hepatitis B (CHB) patients have yet to be clarified. The objective of this study was to analyse the HBV-specific T cell response, in parallel with peripheral and intrahepatic viral parameters, in patients undergoing NA discontinuation. METHODS: Twenty-seven patients without cirrhosis with HBeAg-negative CHB with complete viral suppression (>3 years) were studied prospectively. Intrahepatic HBV-DNA (iHBV-DNA), intrahepatic HBV-RNA (iHBV-RNA), and covalently closed circular DNA (cccDNA) were quantified at baseline. Additionally, serum markers (HBV-DNA, HBsAg, HBV core-related antigen [HBcrAg] and HBV-RNA) and HBV-specific T cell responses were analysed at baseline and longitudinally throughout follow-up. RESULTS: After a median follow-up of 34 months, 22/27 patients (82%) remained off-therapy, of whom 8 patients (30% of the total cohort) lost HBsAg. Baseline HBsAg significantly correlated with iHBV-DNA and iHBV-RNA, and these parameters were lower in patients who lost HBsAg. All patients had similar levels of detectable cccDNA regardless of their clinical outcome. Patients achieving functional cure had baseline HBsAg levels ≤1,000 IU/ml. Similarly, an increased frequency of functional HBV-specific CD8+ T cells at baseline was associated with sustained viral control off treatment. These HBV-specific T cell responses persisted, but did not increase, after treatment withdrawal. A similar, but not statistically significant trend, was observed for HBV-specific CD4+ T cell responses. CONCLUSIONS: Decreased cccDNA transcription and low HBsAg levels are associated with HBsAg loss upon NA discontinuation in patients with HBeAg-negative CHB. The presence of functional HBV-specific T cells at baseline are associated with a successful outcome after treatment withdrawal. LAY SUMMARY: Nucleos(t)ide analogue therapy can be discontinued in a high proportion of chronic hepatitis B patients without cirrhosis. The strength of HBV-specific immune T cell responses may contribute to successful viral control after antiviral treatment interruption. Our comprehensive study provides in-depth data on virological and immunological factors than can help guide individualised therapy in patients with chronic hepatitis B.

Detecting hepatitis B virus in surgical smoke emitted during laparoscopic surgery.

BACKGROUND: Hepatitis B virus (HBV) transmission is known to occur through direct contact with infected blood. There has been some suspicion that the virus can also be detected in aerosol form. However, this has never been directly shown. The purpose of this study was to sample and analyse surgical smoke from laparoscopic surgeries on patients with hepatitis B to determine whether HBV is present. METHODS: A total of 11 patients who underwent laparoscopic or robotic abdominal surgeries between October 2014 and February 2015 at Korea University Anam Hospital were included in this study. A high efficiency collector was used to obtain surgical smoke in the form of hydrosol. The smoke was analysed by using nested PCR. RESULTS: Robotic or laparoscopic colorectal resections were performed in 5 cases, laparoscopic gastrectomies in 3 cases and laparoscopic hepatic wedge resections in another 3 cases. Preoperatively, all of the patients had positive hepatitis B surface antigen (HBsAg). 2 patients had detectable HBsAb, and 2 were positive for hepatitis B e antigen. 3 patients were taking antihepatitis B viral medications at the time of the study. The viral load measured in the patients' blood was undetectable to 1.7×108 IU/mL. HBV was detected in surgical smoke in 10 of the 11 cases. CONCLUSIONS: HBV is detectable in surgical smoke. This study provides preliminary data in the investigation of airborne HBV infection.

Regional and ethnic aspects of viral hepatitis B among pregnant women.

OBJECTIVES: The aim of this study was to determine the prevalence of HBV infection among pregnant women in districts of Eastern Slovakia with a diverse prevalence of Roma population. METHODS: Overall 59,279 serum samples from 9 regional departments of clinical microbiology from Eastern Slovakia were collected in the period from January 2008 till December 2009 and analysed. RESULTS: The number of HBsAg positive samples overall and during pregnancy was 1.74% and 2.12%, respectively. Comparing districts with higher (> 5%) and lower (< 5%) Roma population, there was no significant difference in the prevalence of HBsAg positive samples overall (1.95% vs.1.62%). However, in the subgroup of pregnant women the prevalence of HBsAg positive samples (2.72% vs. 0.95%) differs significantly (p < 0.01). CONCLUSIONS: The prevalence of HBV infection among pregnant women in Eastern Slovakia did not rapidly exceed the estimated nationwide prevalence. However, in districts with higher Roma population the expected higher prevalence of HBV infection was confirmed. This indicates the need to pay special attention to the prevention of hepatitis B in these districts.

Australia antigen: detection and transmission in shellfish.

Australia antigen was found in clams contaminated by drainage of untreated sewage from a coastal hospital. In closed-system aquariums, the antigen was ingested by clams and transmitted to previously uninfected clams. In opensystem aquariums, the titer of Australia antigen decreased with time, suggesting viral concentration rather than replication.

Australia antigen (hepatitis B antigen): a conformational antigen dependent on disulfide bonds.

Reduction and alkylation of purified hepatitis-associated Australia antigen (hepatitis B antigen) resulted in a total loss of serologic activity. The reduced and alkylated protein formed a single band with a sedimentation coefficient of 31S on analytical ultracentrifugation, and no subunits were detected by Sephadex gel filtration. Although this preparation induced a delayed hypersensitivity response when injected into guinea pigs, it failed to stimulate humoral antibody formation. The data suggest that hepatitis B antigen is a conformational antigen critically dependent upon the disulfide bonds of the protein moiety.

High prevalence of hepatitis B-antibody loss and a case report of de novo hepatitis B virus infection in a child after living-donor liver transplantation.

AIM: To assess the seroprevalence of hepatitis B virus (HBV) immunity among previously vaccinated pediatric liver transplant recipients and present a case report of de novo hepatitis B infection after liver transplantation. METHODS: This study focused on children with chronic liver diseases who received primary hepatitis B immunization and had a complete dataset of anti-HBs before and after liver transplantation between May 2001 and June 2017. Medical records were retrospectively reviewed for potential factors relating to HBV immunity loss. RESULTS: In total, 50 children were recruited. The mean time from liver transplantation to anti-HBs testing was 2.53 ± 2.11 years. The mean anti-HBs levels before and after liver transplantation were 584.41 ± 415.45 and 58.56 ± 6.40 IU/L, respectively. The rate of non-immunity (anti-HBs < 10 IU/L) in the participants was 46% (n = 26) at one year, 57% (n = 7) at two years and 82% (n = 17) at > three years following liver transplantation. The potential factors relating to HBV immunity loss after liver transplantation were identified as anti-HBs (P = 0.002), serum albumin (P = 0.04), total bilirubin (P = 0.001) and direct bilirubin (P = 0.003) before liver transplantation. A five-year-old boy with biliary cirrhosis received 4 doses of HBV vaccine with an anti-HBs titer of > 1000 IU/L and underwent liver transplantation; his anti-HBc-negative father was the donor. After liver transplantation, the boy had stenosis of the hepatic artery up to the inferior vena cava anastomosis and underwent venoplasty three times. He also received subcutaneous injections of enoxaparin for 5 mo and 20 transfusions of blood components. Three years and ten months after the liver transplantation, transaminitis was detected with positive tests for HBsAg, HBeAg, and anti-HBc (2169.61, 1706 and 8.45, respectively; cutoff value: < 1.00) and an HBV viral load of 33212320 IU/mL. CONCLUSION: The present study showed that loss of hepatitis B immunity after liver transplantation is unexpectedly common. In our case report, despite high levels of anti-HBs prior to transplantation, infection occurred at a time when, unfortunately, the child had lost immunity to hepatitis B after liver transplantation.

Purification and biophysical characterization of hepatitis B antigen.

Hepatitis B antigen (HB Ag) has been purified from serum by pepsin digestion and equilibrium sedimentation. The sedimentation coefficient, density, particle size, and electrophoretic mobility have been determined before and after purification and found to be unaltered. In addition, the diffusion constant and molecular weight of purified HB Ag have been documented. Because the biophysical characteristics found are not consistant with those of any known virus, it is believed that native HB Ag is made up almost entirely of incomplete virus.

Hepatitis B virus markers in diabetes mellitus.

Serum markers for hepatitis B virus (HBV) were studied in 395 healthy control subjects and in 100 diabetic patients. Of the patients, 28 had type I diabetes, 31 had type II diabetes requiring insulin, and 41 had type II diabetes treated with oral agents or diet alone. None gave history of previous icterus or other signs of hepatitis, had received blood transfusions, or had been on hemodialysis. There was a significant difference in the prevalence of HBV markers (mainly HB surface antibody) between the diabetic group and the controls (51% versus 25%, P less than 0.001). The control subjects included hospital personnel and, hence, their risk of HBV exposure was already relatively high. The increased occurrence of HBV markers did not seem to be related to diabetes duration, patient age, intake of insulin injections, or presence of microvascular complications. This study reveals a high degree of exposure to HBV in a moderately controlled diabetic group and possibly a high degree of proneness to subclinical hepatitis B.

Isolation of hepatitis B surface antigen (HBsAg) by affinity chromatography on antibody-coated immunoadsorbents.

Hepatitis tb surface antigen (HBsAg) was isolated from human serum by two steps of affinity chromatography on antibody-coated gels. HBsAg-positive serum was passed through a column packed with guinea pig anti-HBsAg antibodies covalently bound to CNBr-activated beaded agarose gel. The majority of non-specifically bound proteins was removed by washing the gel with increased concentrations (0.5 M) of NaCl in Tris buffer. Elution of the specifically bound HBsAg was carried out with 3 M NaSCN. Residual normal human serum proteins present in the eluate were removed by passing the partially purified HBsAg through an immunoadsorbent coated with rabbit antibodies directed against human serum proteins. After this treatment normal human serum proteins could no longer be demonstrated by passive hemagglutination in the isolated HBsAg. Cross-reactions between HBsAg and normal human serum proteins could not be demonstrated. Both antibody-coated immunoadsorbents could be used over ten times without significant loss of their binding capacity.

Expression of deletion mutants of the hepatitis B virus protein HBx in E. coli and characterization of their RNA binding activities.

The hepatitis B virus protein HBx has been implicated in the development of liver cancer. It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner. This DNA binding activity might be relevant for HBx oncogene character. To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E. coli. Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA. The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half. AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation. By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide. This new aspect of HBx function is discussed in the context of cellular transformation.

Expression of hepatitis B virus antigens in attenuated Salmonellae for oral immunization.

The aim of our work is to identify hepatitis B virus antigens that can be stably expressed in attenuated Salmonellae and elicit protective immune responses as live oral route vaccines. As a first carrier system, we expressed T-cell and B-cell epitopes of hepatitis B virus as fusion proteins with the non-toxic subunit B (LT-B) in attenuated Salmonellae. These recombinant Salmonellae elicited anti-LT-B T- and B-cell immune responses and anti-HBV nucleocapsid antigen (HBcAg) T-cell responses when fed to mice. To combine the protective potential and the high immunogenicity of HBc with the induction of virus neutralizing antibodies to HBV surface antigen, we constructed vectors expressing hybrid HBc/pre-S particles in which the pre-S epitopes were surface-exposed. With one of these vectors, stable constitutive high level expression of hybrid HBc/pre-S2 particles was achieved in several attenuated Salmonella strains. When recombinant Salmonellae expressing such hybrid HBc/pre-S2 fusion proteins were fed to mice, the animals developed high titres of anti-HBcAg-specific serum IgG after a single or multiple oral immunizations, depending on the strain used as a carrier. In addition, lower titered antibodies against the pre-S2 antibody-binding sites were elicited. This is the first HBV antigen eliciting high-titered immune responses after a single oral immunization in recombinant Salmonellae. The immunogenicity of periplasmic LT-B and cytoplasmic HBc/pre-S2 shows that surface exposure of a foreign antigen is not a prerequisite for its immunogenicity in live attenuated Salmonellae.

Hepatitis B (surface) antigen testing by radioimmunoassaymexperience in a very large volunteer donor population.

The results of hepatitis B surface antigen (HBs-Ag) testing in a large volunteer blood donor population are described. Counterelectrophoresis and three versions of solid-phase radioimmunoassay technic are compared and evaluated. Initial results suggested that the radioimmunoassay technic are compared and evaluated. Initial results suggested that the radioimmunoassay technic detected more than five times as many reactive donors as did counterelectrophoresis. The specificity of the radioimmunoassay technic has been increased by successive modifications, and recent results show that the technic detects 73 percent more reactive donors than does counterelectrophoresis. Not all of these reactions are specific, and it is estimated that the true gain in detection of HBsAg carriers is 49 percent of the value found by counterelectroesis. The incidence of HBsAg carriers in the America Red Cross donor population is about 1.25 per 1,000.

The detection of hepatitis B antigen in hepatic parenchyma by the fluorescent antibody technic.

Tissue sections from 42 specimens of liver were examined by indirect immunofluorescence microscopy for the presence of hepatitis B antigen (HB Ag). In all cases the serologic status of HB Ag was known. Fourteen of the specimens were also examined by electron microscopy. In four biopsies from three patients positive cytoplasmic fluorescence was detected using antisera prepared in animals and 20-nm. nuclear particles were found by electron microscopy. These patients were all seropositive for HB Ag, all had chronic aggressive hepatitis or active cirrhosis, and all were receiving immunosuppressive therapy at the time of examination. Nuclear fluorescent staining was demonstrated when one of these biopsies was re-examined using a human antiserum.

Electron microscopic study of the distribution of the Australia antigen in individual sera of 50 serologically positive blood donors and two patients with serum hepatitis.

The distribution of the morphological types of Australia antigen in 50 blood donors and two patients with serum hepatitis is described.The significance of the high incidence of immune complexes and Dane particles in these persistent carriers is discussed.

An automated passive haemagglutination technique suitable for the detection of hepatitis B virus antigen and antibody in blood donors.

A method is described for preparing purified hepatitis B antigen (HB Ag) viral antigen without density gradient centrifugation. A method for sensitizing human group O red cells with this preparation is given, together with the technical details of an automated passive haemagglutination technique suitable for the mass screening of blood donors for HB virus and anti-HB antibody.

An evaluation of low voltage counterimmunoelectrophoresis for the detection of hepatitis-B antigen (HB Ag).

A newly available low voltage counterimmunoelectrophoresis (CIEP) system for the detection of HB Ag (Hapindex, Ortho Diagnostics) was compared with a conventional CIEP method used at this laboratory. A total of 1216 sera were tested. The Hapindex system was found to be at least as sensitive as the conventional CIEP. No false positives were found in this series. The elimination of any preparative work makes the Hapindex system particularly suitable for laboratories not testing large numbers of sera for HB Ag. It also eliminates many of the contamination hazards inherent in the conventional method.

Orcein staining of hepatitis B antigen in paraffin sections of liver biopsies.

Liver biopsies from 97 hepatitis B antigen (HBsAG)-positive patients were stained by a modified orcein method described by Shikata et al (1974) in order to detect the antigen in liver tissue. The results were consitently negative in acute hepatitis, but positive in nearly two-thirds of biopsies from 53 patients with chronic liver disease. The distribution of positive staining was frequently irregular so that there is a problem of sampling error in needle biopsies. The deposits were seen in the cytoplasm of liver cells and occasionally in Kupffer cells, but never in nuclei. There was an inverse relationship between staining and parenchymal necrosis. Biopsies from asymptomatic HB(s)Ag carriers were often strongly positive, as were "ground-glass" hepatocytes in carriers and patients with chronic liver disease. The mechanism of staining is unclear but may be related to the presence of disulphide bonds in (HBsAG. The technique is simple and of use both in fresh and stored material.

Hepatitis B surface antigen (HBsAg) in the liver of patients with hepatitis; a comparison with serological detection.

Chronic hepatitis was diagnosed on liver biopsy of 76 patients; 52 (68%)had HBsAg. Of the 52 patients with HBsAg, 23% had HBsAg shown by immunofluorescence on the liver, while it could not be detected with radioimmunoassay on the serum; 77% had HBsAg detectable in liver and in serum, and none had HBsAg in serum only. HBsAg was detected more frequently in chronic aggressive hepatitis and active cirrhosis than in chronic persistent hepatitis and cirrhosis with little activity. No correlation was found in the different forms of chronic hepatitis between the HBsAg status on the one hand, and levels of transaminases, gammaglobulins, and auto-antibodies on the other. Acute hepatitis was diagnosed on liver biopsy of 24 patients; 50% had HBsAg. Liver tissue positivity was very low in the fully developed stage compared to serum positivity. In 146 patients with other liver ailments, both liver and serum were negative for HBsAg.

The hepatitis B virus as a molecular model for chronic infection: synthesis of hepatitis B surface and e antigens in mouse L cells transfected with closed circular viral DNA.

Linear hepatitis B virus (HBV) DNA, excised from a recombinant plasmid with EcoR1, was purified by preparative electrophoresis on agarose gels and incubated with phage T4 ligase to form either monomeric or dimeric closed circles. Thymidine kinase deficient mouse L cells were cotransfected with thymidine kinase (tk) and circular HBV DNAs and grown in hypoxanthine medium. Colonies of tk-transformed cells, selected after 3-4 weeks of incubation and subcultured in HAT medium, synthesized either hepatitis B surface antigen (HBsAg) alone or HBsAg in combination with hepatitis B e antigen (HBeAg). The various cell colonies differed in plating efficiency, growth rates, cellular appearance, and extent of viral antigen synthesis. Southern hybridization analysis showed the presence of HBV-related sequences in high molecular weight DNA prepared from cells expressing viral antigens. Digestion of cellular DNAs with restriction endonucleases indicated integration of the entire viral genome.

Can the blood-transmitted hepatitis problem be solved?

Of the approaches presently available for prevention of blood-transmitted hepatitis, most clearly effective is the avoidance of high-risk donor populations, such as most paid commercial donors. It should be emphasized that even after screening for hepatitis B antigen most paid donor blood carries with it a five-ten times higher risk of transmitting hepatitis than volunteer donor blood. Hepatitis B screening is certainly the next most useful approach. However, it has been pointed out that even with the most sensitive of current assays, as much as two-thirds of infective blood may not be eliminated. In part this is due to failure to detect hepatitis B antige, which may be detectable by such currently investigational approaches as selective antibody inhibition or testing for Core antibody. However, a major factor is the inability of hepatitis B screening to eliminate all infectious blood appears to be due to the existence of viruses other than hepatitis B that play a major role in blood-transmitted hepatitis. Tests for detection of carriers of this virus, or viruses, remain to be developed. Improved reporting of hepatitis cases with investigation and registration of implicated donors may provide an additional means for reducing the incidence of hepatitis transmission. The efficiency of additonal approaches, such as transaminase screening of donors and passive or active immunization, remains to be determined.