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Approximately 70% of kidney transplant recipients are non-responders to conventional hepatitis B virus (HBV) vaccines. We examined whether Fendrix™, an HBV vaccine containing 3-O-desacyl-4'-monophosphoryl lipid A (MPL) as adjuvant, could induce HBV immunity in these patients and compared their vaccination efficacy with healthy controls tested previously by the same assays. We selected 35 kidney transplant recipients who had been vaccinated at least thrice against HBV but had never displayed anti-HBs antibodies. We re-assessed their anti-HBs antibody titres and further determined cellular HBV immunity by proliferation assay and interferon (IFN)-γ ELISpot. Seventeen recipients did neither display humoral nor cellular immunity and could be tested prior to and at month 1 after vaccination. Of note, HLA antigens associated with non-response to HBV vaccination (HLA-DRB1*03 and HLA-DQB1*02) were over-represented in these 17 recipients. At month 1 after a single vaccination with Fendrix™, we observed a significant increase in anti-HBs antibodies (P = 0.02). In seven of 17 recipients, we detected anti-HBs antibodies ≥10 IU/l (10-264), in four HBV-specific lymphocyte proliferation (stimulation index of 2.6-8.7) and in one specific IFN-γ responses (12 spots increment). The vaccination response to Fendrix™ was significantly higher (P = 0.035) than the response to HBVaxPro™ in young healthy controls. In summary, the results show that a single vaccination with Fendrix ™ could already induce HBV-specific humoral and/or cellular responses in ten of 17 kidney transplant patients. Thus, Fendrix™ appears as an efficient vaccine in this patient cohort., (© 2016 The Foundation for the Scandinavian Journal of Immunology.)

Non-responsiveness to conventional hepatitis B vaccines in individuals at high risk of exposure to hepatitis B virus (HBV) is an important public health problem and of particular relevance in health care providers. Yeast-derived conventional HBsAg vaccines fail to induce protective antibody titers in up to 10% of immune competent vaccinees. Therefore, a third generation HBV vaccine, Sci-B-Vac™, was developed which contains in addition to the small S antigen the PreS1 and PreS2 antigens. This vaccine proved to induce a highly potent cellular and humoral immune response in healthy individuals as well as protective antibody levels in non- and low-responders to conventional HBV vaccines. The aim of the study was to examine whether Sci-B-Vac™ triggers cellular and humoral immunity in individuals who failed immunization with conventional vaccines. We immunized 21 volunteers (15 non- and 6 low-responders) according to the standard vaccination schedule (0, 4 and 24 weeks), determined the cellular immunity by proliferation assay and interferon (IFN)-γ ELISpot and measured the anti-HBs antibody titers prior to each vaccination and four weeks after the third vaccine dose. Following three vaccinations, PreS/S-specific T-cell proliferation was detected in 8 out of 15 non-responders and 5 out of 6 low-responders. Specific IFN-γ responses were measured in 2 out of 15 non-responders and 4 out of 6 low-responders. All but one (20/21) study participants developed anti-HBs titers ≥10IU/l after three vaccinations. Anti-HBs ≥100IU/L were detected in 12 out of 15 non-responders and in 6 out of 6 low-responders. Anti-HBs ≥10IU/l and <100IU/l were found in 2 non-responders. These results indicate that Sci-B-Vac™ induces cellular immunity as well as protective anti-HBs antibody titers in non- and low-responders. In conclusion, these results confirm that Sci-B-Vac™ should be administered to non-responders to conventional HBV vaccines and patients with impaired immune function., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: The fully automated and closed LIAISON(®)XL platform was developed for reliable detection of infection markers like hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis C virus (HCV) antibodies (Ab) or human immunodeficiency virus (HIV)-Ag/Ab. To date, less is known about the diagnostic performance of this system in direct comparison to the common Abbott ARCHITECT(®) platform., Objectives: We compared the diagnostic performance and usability of the DiaSorin LIAISON(®)XL with the commonly used Abbott ARCHITECT(®) system., Study Design: The qualitative performance of the above mentioned assays was compared in about 500 sera. Quantitative tests were performed for HBsAg-positive samples from patients under therapy (n=289) and in vitro expressed mutants (n=37). For HCV-Ab, a total number of 155 selected samples from patients chronically infected with different HCV genotypes were tested., Results: The concordance between both systems was 99.4% for HBsAg, 98.81% for HCV-Ab, and 99.6% for HIV-Ab/Ag. The quantitative LIAISON(®)XL murex HBsAg assay detected all mutants in comparable amounts to the HBsAg wild type and yielded highly reliable HBsAg kinetics in patients treated with antiviral drugs. Dilution experiments using the 2nd International Standard for HBsAg (WHO) showed a high accuracy of this test. HCV-Ab from patients infected with genotypes 1-3 were equally detected in both systems. Interestingly, S/CO levels of HCV-Ab from patients infected with genotype 3 seem to be relatively low using both systems., Conclusions: The LIAISON(®)XL platform proved to be an excellent system for diagnostics of HBV, HCV, and HIV with equal performance compared to the ARCHITECT(®) system., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8(+) and CD4(+) T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven.

Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8(+) T-cell response than with the previously used vaccines. Using multicolor flow cytometry and an in vivo cytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.

The woodchuck together with the woodchuck hepatitis virus (WHV) is an excellent model to study the pathogenesis of hepadnaviral infections. Chronic WHV infection causes severe liver disease and hepatocellular carcinoma in woodchucks. The mechanism of viral clearance is not fully understood, interferons seem to play a major role in down-regulating viral replication prior to elimination of infected hepatocytes. We investigated on the pattern of cytokine and T-cell-marker expression in livers of woodchucks chronically infected with WHV. RNase-protection-assay (RPA) was used to determine mRNA of woodchuck specific genes (TNF-alpha, IFN-gamma, IL-15, CD3, CD4, CD8). Serial liver biopsies were performed daily or weekly in eight chronic WHV-carrier woodchucks. Cytokine/T-cell-marker expression differed significantly between the time points up to +/-50% within each woodchuck. The different expression patterns of cytokines or T-cell-markers did not correlate to the (weak) fluctuations in the viremia but may explain the observed fluctuations in the WHV/HBV-load in chronically infected individuals. Furthermore, we observed associations between cytokine and T-cell-marker expression. The marginal fluctuations in viremia during the chronic infection may indicate, that, once the chronic hepadnaviral infection is established, cytokines/interferons expressed endogenously (i.e. not vector-borne or injected) play only a minor role.

Background: Previous data indicate that a transfer of specific humoral and cellular immunity by way of allogeneic hematopoietic cell transplantation (HCT) should, in principle, be possible., Methods: In the HCT setting with a follow-up of up to 55 months, we studied the transfer of hepatitis B virus (HBV) specific immunity from electively immunized donors into HLA compatible recipients suffering from chronic myeloid leukemia (CML). After excluding preexisting HBV specific immunity in donor-recipient pairs, 27 prospective donors were vaccinated against HBV. In addition, on an average of 22 months postHCT, 8 of the 19 recipients were immunized once for HBV., Results: Donor vaccination resulted in detectable hepatitis B surface (HBs) antibodies in 85% of donors and specific cellular in vitro responses in 77%. Two weeks postHCT, 86 and 67% of the recipients displayed positive humoral and cellular HBV reactions, respectively, which then decreased. Afterwards, HBV immunity reappeared in 83% of the recipients without revaccination. Following a single vaccination in recipients, seven of eight displayed a typical memory response. An HBV specific response was already detectable 1 week after vaccination, approximately 1,300-fold (humoral) and 60-fold (cellular) higher than observed in the corresponding donors after a single immunization., Conclusions: The "spontaneous" recurrence of HBV immunity and the memory response in recipients give evidence for an elective immune transfer (e.g., for viral antigens) by way of allogeneic HCT.

Background: Reinfection of liver grafts with hepatitis B virus (HBV) is a pertinent problem in clinical liver transplantation, requiring the development of new treatment strategies. However, no animal model is currently available to study the course and mechanism of hepatitis B reinfection. This was the reason to establish the technique of liver transplantation in the woodchuck, which is a widely used animal model for HBV infection., Material and Methods: For the reinfection study, woodchuck hepatitis virus (WHV)-negative animals were selected as donors, whereas chronic carriers served as recipients (n=3). Immunosuppression consisted of cyclosporine in a daily dose of 5 mg/kg. Blood and liver samples were obtained before and 8 hr, 3 weeks, and 10 weeks after transplantation. Virological markers included serological testing for WHV DNA, WHV surface antigen (WHsAg), core antigen (WHcAg), and their antibodies (anti-WHs and anti-WHc). WHV DNA replication intermediates and viral RNA were detected by Southern blot hybridization and Northern blot, respectively. Viral proteins in the liver were visualized by immunohistochemistry for WHsAg and WHcAg., Results: Early after transplantation membranous but no intracytoplasmic staining for WHsAg was detected in the liver graft, which was negative for WHcAg as well as WHV-DNA and RNA. Nearly all hepatocytes in the liver grafts of animals killed at 3 weeks and 10 weeks posttransplant showed strong membranous (WHsAg) and intracytoplasmic (WHsAg and WHcAg) staining, which was higher in frequency and intensity than in carriers before transplantation. The apparently reduced level of WHV replication intermediates and viral RNA in the reinfected liver grafts compared with the carrier animals was caused by the severe morphological changes leading to a replacement of hepatocytes by extended portal infiltrates., Conclusion: The woodchuck proved to be a suitable model to study WHV reinfection after liver transplantation, because the operative procedure was well tolerated. The first sign of viral presence in the graft was WHsAg detected exclusively in the sinusoids. Reinfection was proven by heavy intracytoplasmic staining for WHsAg and WHcAg in the majority of hepatocytes and detection of viral DNA and RNA in the graft.

Adoptive transfer of immunity to hepatitis B virus (HBV) is not provoked solely by bone marrow, but also by liver transplantation, although transiently. In the current study, simultaneous bone marrow transplantation, which possibly can increase the number of antibody-secreting cells, was performed to augment the efficacy of transferring HBV immunity. Stimulation of donor-derived immune cells by postoperative vaccination was used to investigate whether a secondary immune response can be induced in recipients. Orthotopic liver transplantation (n = 28), performed in three rat strain combinations representing different genetic constellations, was compared with bone marrow-augmented liver transplantation (n = 21). Donors had been vaccinated twice with recombinant hepatitis B surface antigen (HBsAg). Recipients surviving more than 10 weeks received a boost vaccination. All animals were monitored weekly for the presence of antibodies to HBsAg (anti-HBs). Effective anti-HBs titers were measured in 82% of liver recipients (23 of 28 recipients) and lasted from 2 to 9 weeks. Ninety percent of bone marrow-augmented liver recipients (19 of 21 recipients) seroconverted, with anti-HBs persisting from 2 to 12 weeks. A greater seroconversion rate, prolonged titer duration, and different pattern of titer development were observed in bone marrow-augmented liver recipients, although statistical significance could not be obtained because of the small numbers of comparable animals. Posttransplantation vaccination in recipients of combined grafts did not arouse a typical secondary antibody response, but showed a tendency toward an earlier and stronger response to vaccine in comparison to recipients without immune transfer. Simultaneous bone marrow transplantation showed an augmenting, but limited, effect on humoral immune transfer. Therefore, other potentially promising cellular strategies, such as transfer of in vivo and ex vivo stimulated antigen-specific cells should be pursued further. Improvement of the effect of postoperative vaccination possibly can be achieved by optimizing the immunization protocol.

It has been recently shown that Toll-like receptor ( TLR) signalling in murine nonparenchymal liver cells ( NPCs) is suppressed in the presence of Hepatitis B virus surface antigen ( HBs Ag). It is not clear, however, whether this is also relevant for the adaptive immune responses and how this effect is mediated. Peripheral blood mononuclear cells ( PBMCs) from Hepatitis B virus ( HBV) patients and controls were stimulated by TLR ligands in the absence or presence of autologous serum. Interestingly, TLR-mediated cytokine expression ( Interleukin-6 and -10) as well as TLR3-induced interferon ( IFN) expression in PBMCs of HBV patients was significantly higher than in the healthy volunteers, showing a negative correlation with the levels of HBs Ag. In addition, TLR3-mediated IFN-γ production was inhibited in the presence of HBV-containing serum. To mechanistically analyse this observation, murine Kupffer cells ( KCs) and sinusoidal endothelial cells ( LSECs) were stimulated with TLR3 ligands in the presence or absence of HBs Ag. Mixed lymphocyte reactions were performed to study T-cell activation induced by TLR-stimulated NPCs. Gene expression of cytokines and TLR3 was analysed by quantitative rt- PCR, and activation of transcription factors was assessed by Western blot or reporter gene assays. TLR-induced expression of interferon γ, interferon sensitive genes and proinflammatory cytokines in murine KCs and LSECs was efficiently suppressed in the presence of HBs Ag, whereas the expression of anti-inflammatory cytokines was enhanced. Activation of NFκ B, IRF-3 and MAPKs in these liver cells was potently suppressed by HBs Ag. T-cell activation mediated through TLR3-stimulated KCs or LSECs was suppressed by HBs Ag which could be reverted by anti- IL-10 antibodies. These findings may, at least in part, explain how HBV evades innate and adaptive immune responses to maintain a persistent infection. [ABSTRACT FROM AUTHOR]