Creation of an immunodeficient HLA-transgenic mouse (HUMAMICE) and functional validation of human immunity after transfer of HLA-matched human cells.

Research on human immunology has been hindered by the lack of optimal small animal models, given that the protective immune responses of human and non-human species show significant differences. However, due to ethical constraints[1] and the high cost of clinical trials, it is urgent to improve the current animal models that can mimic faithfully human physiology, particularly the human immune system (HIS). HIS mice had been generated recently by engrafting human hematopoietic stem cells (hHSCs) or human peripheral mononuclear cells (hPBMCs) into highly immuno-deficient mice such as NSG, NOG or NRG mice. However, a major experimental drawback for studies using these models is the rapid onset of Graft-versus-Host Disease (GvHD). In the present study, we overcome this limitation by generating new immuno-deficient mice named "HUMAMICE" (HLA-A2+/+/DR1+/+/H-2-ß2m-/-/IAß-/-/Rag2-/-/IL2rγ-/-/Perf-/- mice), which expressed human HLA molecules instead of mouse MHC molecules (H-2), and whose immuno-deficient status was reversed by transferring functional HLA-matched PBMCs thus producing mice with an immuno-competent status with a functional human immune system. We showed that in this HLA-matched context, the hPBMC-transfer led to high lymphocytes engraftment rates without GvHD over three months in this novel mouse model. Furthermore, to evaluate the utility of the hPBMC-HUMAMICE, we immunized them with commercial vaccine of Hepatitis B virus (HBsAg, Hepvac@) which resulted in robust and reproducible production of high levels of HBsAg-specific antibodies, implying that both transferred T and B lymphocytes were functional in HUMAMICE. These responses are comparable to those observed in human clinical trials with this identical vaccine. In conclusion, these findings indicated that the HLA-matched-hPBMC-HUMAMICE represents a promising model for dissecting human immune responses in various human diseases, including infectious diseases, cancers and tumors, and to facilitate the development of novel vaccines and cellular therapies.

High risk of occult hepatitis B virus infection in leukemia patients from China.

In this study, we assessed the prevalence of overt and occult hepatitis B virus (HBV) infection among leukemia patients. Among 256 leukemia patients and 377 fracture patients (control group), we found that the hepatitis B surface-antigen-positive rate was greater in leukemia patients than in the controls (odds ratio, 2.08; p = 0.01). Moreover, the prevalence of occult HBV infection was higher in leukemia patients than in the controls (10.5 % vs. 2.9 %; odds ratio, 3.92; p < 0.001). The HBV genotype distribution differed significantly between the leukemia and chronic hepatitis B or control groups (p < 0.001 and 0.01, respectively); specifically, genotype C was primarily observed in occult HBV infection patients with leukemia. The stop codon mutation rate or amino acid substitutions in the major hydrophilic region did not differ between the groups. Thus, the prevalence of occult hepatitis B is higher in leukemia patients, and the HBV genotype distribution differs between patients with leukemia and chronic hepatitis B virus infection.

Influence of single nucleotide polymorphisms of cytokine genes on anti-HBs antibody production after hepatitis B vaccination in a Japanese young adult population.

Hepatitis B (HB) vaccination is one of the most efficient tools to prevent the transmission of the virus. Considerable variability exists in HB vaccine responses, with 5-10% of healthy Japanese adults demonstrating no response following a standard vaccination. Recently, polymorphisms of immune-regulatory genes, such as cytokine genes, have been reported to influence the immune response to HB vaccine. The aim of this study was to investigate the underlying mechanisms of the genetic association between several cytokine gene polymorphisms and the immune response to HB vaccination in a Japanese population. One hundred and twenty three vaccinated young adults were classified according to the level of antibody-titer (anti-HBs). Single nucleotide polymorphism typing for IFN-γ (+874, 3'-UTR), IL-10 (-591, -819, -1082), and TNF-α (-308, -857), was accomplished using the PCR-RFLP or SSP-PCR method. The TNF-α (-857) CC type and the IL-10 (-1082) AG type were present more frequently in the low titer group than in the high titer group. The TNF-α (-857) CC type was found to be significantly associated with low response of serum anti-HBs. The anti-HBs antibody was not readily produced in the IL-10 (-1082) AG and TNF-α (-857) CC haplotype. Conversely, the antibody was readily produced in the IL-10 (-1082) AA and TNF-α (-857) CC haplotype, and the IL-10 (-1082) AA and TNF-α (-857) CT haplotype, suggesting a high likelihood of the IL-10 (-1082) AG type to be included in the low anti-HBs group, and high anti-HBs antibody production in those with the TNF-α (-857) CT type. These SNPs may produce ethnically-specific differences in the immune response to HB vaccine in the Japanese population. J. Med. Invest. 63: 256-261, August, 2016.

Respuesta inmunitaria a la vacuna de la hepatitis B en pacientes hemodializados en Brasil y sus factores asociados / Factors associated with the immune response to hepatitis B vaccine in Brazilian hemodialysis patients

Background:Patients on chronic hemodialysis have a lower immune response to vaccination against hepatitis B virus (HBV) than the general population. Aim: To identify factors that may interfere with immunization against hepatitis B virus (HBV) in Brazilian hemodialysis patients and analyze the evolution of the level of antibodies. Patients and Methods: A retrospective longitudinal study, using records of patients on hemodialysis in the years 2000-2008. Non-responder patients, defined as a level of anti-HBs less than 10 IU/mL, were identified. The effect of social and demographic factors, clinical and laboratory data on the lack of response was evaluated. Logistic regression analysis was used to assess the independent effect of each factor. The difference between initial and final anti-HBs levels (24 months), was also analyzed. Results: Fifty seven percent of patients responded adequately to vaccination. After adjustment with other variables, the only factor associated with immune response was serum ferritin. Responding patients of less than 40 years of age did not have a significant decrease in antibody titers over time. The initial anti-HBs title, influenced the final title. Fifty percent of non-responders achieved serum levels of protection after revaccination. Conclusions: The study showed that ferritin may be a marker of reduced immune response. Patients aged less than 40 years were the only ones who maintained over time their initial anti-HBs levels.

Transfer of HBV genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice.

Studies of mechanisms responsible for the persistence of hepatitis B virus (HBV) infection have been hindered by a lack of appropriate animal models. HBV genomes can be delivered to livers of mice using hydrodynamic injection or high doses of an adenoviral vector; these lead to clearance of HBV. We found that infection of immunocompetent mice with low doses of an adenoviral vector resulted in persistent HBV infection; the mice neither underwent seroconversion to production of antibodies against HBV nor developed a strong HBV-specific effector T-cell response. As in patients with chronic HBV infection, DNA vaccination failed to generate T cells that cleared infection. This model of persistent HBV infection could be used to study the pathogenesis of chronic HBV infection and develop new therapeutic strategies.

Differential N-glycosylation of a monoclonal antibody expressed in tobacco leaves with and without endoplasmic reticulum retention signal apparently induces similar in vivo stability in mice.

Plant cells are able to perform most of the post-translational modifications that are required by recombinant proteins to achieve adequate bioactivity and pharmacokinetics. However, regarding N-glycosylation the processing of plant N-glycans in the Golgi apparatus displays major differences when compared with that of mammalian cells. These differences in N-glycosylation are expected to influence serum clearance rate of plant-derived monoclonal antibodies. The monoclonal antibody against the hepatitis B virus surface antigen expressed in Nicotiana tabacum leaves without KDEL endoplasmic reticulum (ER) retention signal (CB.Hep1(-)KDEL) and with a KDEL (Lys-Asp-Glu-Leu) fused to both IgG light and heavy chains (CB.Hep1(+)KDEL) were tested for in vivo stability in mice. Full characterization of N-glycosylation and aggregate formation in each monoclonal antibody batch was determined. The mouse counterpart (CB.Hep1) was used as control. Both (CB.Hep1(-)KDEL) and (CB.Hep1(+)KDEL) showed a faster initial clearance rate (first 24 h) compared with the analogous murine antibody while the terminal phase was similar in the three antibodies. Despite the differences between CB.Hep1(+)KDEL and CB.Hep1(-)KDEL N-glycans, the in vivo elimination in mice was indistinguishable from each other and higher than the murine monoclonal antibody. Molecular modelling confirmed that N-glycans linked to plantibodies were oriented away from the interdomain region, increasing the accessibility of the potential glycan epitopes by glycoprotein receptors that might be responsible for the difference in stability of these molecules.

miRNA-mediated silencing in hepatocytes can increase adaptive immune responses to adenovirus vector-delivered transgenic antigens.

Adenovirus vectors based on human serotype 5 can induce potent CD8 T cell responses to vector-encoded transgenic antigens. However, the individual contribution of different cell types expressing antigen upon adenovirus vector injection to the generation of antigen-directed adaptive immune responses is poorly understood so far. We investigated the role of hepatocytes, skeletal muscle, and hematopoietic cells for the induction of cellular and humoral immune responses by miRNA-mediated tissue-specific silencing of antigen expression. Using hepatitis B small surface antigen (HBsAg) as the vector-encoded transgene we show that adenovirus vector dissemination from an intramuscular (i.m.) injection site into the liver followed by HBsAg expression in hepatocytes can limit early priming of CD8 T cells and the generation of anti-HBsAg antibody responses. However, hepatocyte-specific miRNA122a-mediated silencing of HBsAg expression overcame these limitations. Early clonal expansion of K(b)/S(190-197)-specific CD8 T cells was significantly enhanced and improved polyfunctionality of CD8 T cells was found. Furthermore, miRNA122a-mediated antigen silencing induced significantly higher anti-HBsAg antibody titers allowing an up to 100-fold vector dose reduction. These results indicate that miRNA-mediated regulation of antigen expression in the context of adenovirus vectors can significantly improve transgene product-directed immune responses. This finding could be of interest for future adenovirus vaccine vector development.

Switched memory B cells maintain specific memory independently of serum antibodies: the hepatitis B example.

The immunogenicity of a vaccine is conventionally measured through the level of serum Abs early after immunization, but to ensure protection specific Abs should be maintained long after primary vaccination. For hepatitis B, protective levels often decline over time, but breakthrough infections do not seem to occur. The aim of this study was to demonstrate whether, after hepatitis B vaccination, B-cell memory persists even when serum Abs decline. We compared the frequency of anti-hepatitis-specific memory B cells that remain in the blood of 99 children five years after priming with Infanrix -hexa (GlaxoSmithKline) (n=34) or with Hexavac (Sanofi Pasteur MSD) (n=65). These two vaccines differ in their ability to generate protective levels of IgG. Children with serum Abs under the protective level, <10 mIU/mL, received a booster dose of hepatitis B vaccine, and memory B cells and serum Abs were measured 2 wk later. We found that specific memory B cells had a similar frequency in all children independently of primary vaccine. Booster injection resulted in the increase of memory B cell frequencies (from 11.3 in 10(6) cells to 28.2 in 10(6) cells, p<0.01) and serum Abs (geometric mean concentration, GMC from 2.9 to 284 mIU/mL), demonstrating that circulating memory B cells effectively respond to Ag challenge even when specific Abs fall under the protective threshold.

Prolonged stability of the plantibody HB-01 directed against the hepatitis B surface antigen in cryo-preserved tobacco leaves.

Since 1983, several recombinant antibodies have been expressed in important agronomic plant species. However, to date no evaluation has been published about prolonged antibody stability within plant tissues under cryo-preservation conditions. This current report presents an approach to the KDEL-plantibody HB-01 (PHB-01) stability in frozen tobacco leaves by presenting scientific evidence about the stability of a plantibody to a prolonged low temperature exposure in this biological source. Results clearly show that the PHB-01 amount is maintained during the storage of tobacco leaves at -20 degrees C for 90 days. The PHB-01 recovery was not affected by any irreversible physical and/or chemical change produced in tobacco leaves after this cryo-preservation time. The amount of total soluble proteins in the clarified extract decreased in proportion with the storage time and the PHB-01 molecules isolated from frozen leaf extracts were highly pure, >95%, according to an SDS-PAGE assessment under reducing conditions. Low temperature exposure of tobacco leaves did not reveal visible changes in frozen leaves, which is essential for the further extractability of proteins. The PHB-01 is stable in tobacco leaves at -20 degrees C during 90 days, which offers the possibility to overcome problems associated with detrimental climate conditions and optimize purification capabilities.

Particle size influences the immune response produced by hepatitis B vaccine formulated in inhalable particles.

PURPOSE: To test the hypothesis that particle size influences the magnitude of immune response produced by hepatitis B surface antigen (HBsAg) encapsulated in poly (lactic-co-glycolic acid) (PLGA) microspheres. METHODS: Microspheres were prepared by a double-emulsion-solvent-evaporation method, and the particles were characterized for size, morphology, porosity and antigen content. Immunogenicity of encapsulated antigen and safety were studied in rats. Uptake of fluorescent-labeled particles by rat alveolar macrophages was studied by confocal microscopy. RESULTS: With increasing internal aqueous phase (IAP) volume of the microsphere, an increase in particle size and a decrease in particle density were observed. Particles with varying geometric diameters showed aerodynamic diameters between 1 and 6 mu. Addition of poly vinyl alcohol to the IAP resulted in particles with a porous surface. The integrity of HBsAg was maintained upon encapsulation in microspheres. Continuous release of the antigen was observed for formulations incubated in phosphate-buffered saline for 28 days. Immunogenicity increased as a function of particle size upon pulmonary administration. HBsAg encapsulated in approximately 5 mum particles elicited a significantly higher immune response compared to that encapsulated in approximately 12 mum particles. Similar to in vivo immune response data, fluorescent-labeled microspheres of smaller size were taken up more efficiently by rat alveolar macrophages compared to those of larger size. No significant increase in either tumor necrosis factor alpha level in bronchoalveolar lavage fluid or wet lung weight, indicators of inflammation, was observed in rats that received optimized formulations via the pulmonary route. CONCLUSIONS: HBsAg delivered in PLGA microspheres elicited an increase in immunogenicity, and the magnitude of immune response was more pronounced with smaller particles. Inhalable particles of HBsAg could be a viable approach to needle-free vaccination.

An anti-preS2 antibody protects human hepatocytes from hepatitis B virus infection.

BACKGROUND: Hepatitis B virus infection is a major problem in liver transplantation. In this study, we examined the potential efficay of a recombinant adenovirus expressing an antibody against the HBV preS2 antigen (Ab-H-HBV-S2) in preventing HBV recurrent infection after liver transplantation. METHODS: A gene for humanized antibody against the HBV preS2 antigen was cloned into pDC315, a type 5 adenoviral shuttle plasmid. Recombinant virus was obtained by homologous recombination in the 293 packaging cells. The virus containing the Ab-H-HBV-S2 gene was transduced into the rat liver graft during cold preservation. The recombinant virus produced antibody and showed protective effects on human hepatocytes from hepatitis B virus infection in vitro. RESULTS: The recombinant virus titer determined by TCID50 analysis was 5.1x10(10) PFU/mL. The concentration of preS2 antibody in BALB/C nude mice was 16.7 +/- 10.5 microg/mL on day 3,30.9 +/- 13.6 pg/mL on day 7, and lasted for 5 weeks after the injection. At a concentration of 0.5 microg/mL or above, the preS2 antibody protected cultured human hepatocytes from hepatitis B virus. CONCLUSIONS: Adenovirus-mediated gene transduction of anti-preS2 antibody in the transplanted liver may be a useful approach to prevent hepatitis B infection after liver transplantation.

[Natural history of chronic hepatitis B virus infection]. / Historia natural de la infección cronica por el virus hepatitis B.

UNLABELLED: In the last years notable steps have been done towards the understanding of the biology of Hepatitis B Virus (HBV), its natural history and immunopathogenesis, while succesful universal vaccination programs were implemented around the world and important advances in antiviral therapeuthics occurred. Nevertheless, HBV infection remains a public health problem with nearly 350 million carriers worlwide. The natural history of chronic hepatitis B and the spectrum of its clinical forms are complex and variable.We review the natural history of chronic HBV infection describing the early replicative phase and late or non-replicative (inactive carrier) in those patients who adquired the infection during adulthood and the immune tolerant phase, immune clearance and non-replicative in those who acquired the infection in the perinatal period. Emphasis is made in the course of HBeAg negative chronic hepatitis and occult hepatitis B. The complexity of the natural history of hepatitis B depends on viral features, hepatocyte behavior and patient immune response. The intrinsic and extrinsic HBV factors associated with the progression to cirrhosis and hepatocellular carcinoma are also reviewed. KEYWORDS: Hepatitis B, Natural history, liver cirrhosis, hepatocellular carcinoma.

Production of recombinant woodchuck IFNalpha and development of monoclonal antibodies.

Interferon alpha (IFNalpha) is the first line treatment for chronic hepatitis B and C. In order to test new IFNalpha delivery systems and investigate the function of this cytokine in the woodchuck model, the best animal model of chronic hepatitis B, we produced and purified recombinant woodchuck IFNalpha and used it to produce monoclonal antibodies. wIFNalpha5 was cloned in a prokaryotic expression system, expressed as His-tagged protein and then purified. The rwIFNalpha5 protein was found to induce STAT-3 phosphorylation, to enhance 2',5'-oligoadenylate synthetase mRNA levels and to possess a potent antiviral activity. Two monoclonal antibodies were obtained through immunization of rats with rwIFNalpha5. Both recognized rwIFNalpha5 in western blot analysis and one was able to neutralize the antiviral activity of the rwIFNalpha5 and lymphoblastoid IFNalpha preparations. Finally, a capture rwIFNalpha5 ELISA was developed using both antibodies. In summary, the tools generated in this study will allow different forms of IFNalpha delivery as well as different combination therapies in woodchuck hepatitis virus infection to be tested, thus providing useful information for the design of new strategies to treat chronic hepatitis B in humans.

Feasibility study of inhaled hepatitis B vaccine formulated with tetradecylmaltoside.

This study was designed to test the hypothesis that formulation of hepatitis B vaccine with tetradecyl-beta-maltoside (TDM) enhances the immune response after pulmonary administration in a rodent model. Commercially available recombinant hepatitis B vaccine (rHBV) was formulated with varying concentrations of TDM and administered intratracheally to anesthetized male Sprague-Dawley rats. rHBV administered intramuscularly at doses of 2 and 4 microg served as positive controls. All formulations were administered on days 0 and 14 and the immune response was evaluated for 28 days. Specific antibodies generated to HBsAg were analyzed by ELISA. Safety studies were carried out by measuring the levels of alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage (BAL) fluid. There was a significant increase in the immune response when the vaccine was administered intramuscularly at a dose of 4 microg. Only a modest increase in the immune response was observed when plain rHBV was administered intratracheally at the same dose. However, a pulmonary formulation of 4 microg rHBV plus 0.5% TDM produced a fourfold increase in the immune response compared to plain rHBV administered via the pulmonary route. No increase in immune response was observed for formulations containing rHBV plus 0.125% or 0.25% TDM. The levels of ALP and LDH in the BAL fluid suggest that the hepatitis B vaccine plus TDM formulations cause some injury to the lungs after the first intratracheal instillation of the formulation; however, the enzyme levels tended to be lower after the second instillation. The level of TNF-alpha in the BAL fluid of TDM-treated rats was substantially lower than that in rats treated with the positive control substance, sodium dodecyl sulfate. Overall, rHBV formulated with TDM increases the immune response after pulmonary administration, and pulmonary formulation of rHBV plus TDM could be used as an alternative to needle-based delivery of hepatitis B vaccine.

Antibody detection and kinetics of antibody production during early stages of immunization with hepatitis B virus vaccine.

Antibodies to influenza virus and human immunodeficiency virus are detectable in B cells during the early stages of the immune response, prior to their occurrence in plasma. To investigate similar phenomena in a model of immunization against hepatitis B virus (HBV) infection, medical students in Ghana were screened for HBV markers, HBV surface (HBs) antigen (HBsAg), and HBV core antibodies (anti-HBc). Consenting volunteers, 24 of whom were seronegative (susceptible) and 2 of whom were positive for anti-HBc (prior infection), were vaccinated on day 0, day 40, and 6 months. Two sets of 10 blood samples, sequentially collected at intervals of 2 days following each immunization on days 0 and 40, were processed into B-cell lysates and plasma. Solid-phase HBsAg coated on microtiter plates for enzyme immunoassay or nitrocellulose membranes for dot blot assay was used to detect anti-HBs activity by an indirect antiglobulin assay. A commercially procured sandwich immunoassay was used, along with an enzyme-linked immunosorbent assay and a dot blot assay, for the detection of anti-HBs in B-cell lysates and plasma. Following the first injection of vaccine, a single sample of B-cell lysate collected between 5 and 21 days revealed anti-HBs in 18/21 subjects with no plasma antibodies detectable by sandwich immunoassay. After the booster dose was injected on day 40, a single sample of B-cell lysate collected between 44 and 49 days showed anti-HBs in 16/19 subjects, and this was accompanied by plasma antibodies in 8 subjects. In contrast, between 8 and 13 days, both subjects with prior HBV infection showed anti-HBs in B-cell lysates and plasma. Thus, primary immunization with the HBV vaccine appears to transiently elicit low-affinity anti-HBs in B-cell lysates into plasma.

Generation of chimeric HBc proteins with epitopes in E.coli: formation of virus-like particles and a potent inducer of antigen-specific cytotoxic immune response and anti-tumor effect in vivo.

The major aim of the project was to develop the virus-like particles (VLPs) displaying single or multi-epitope of hepatocellular carcinomas (HCC) in Escherichia coli and to evaluate the effect on inducing Ag-specific CD8(+) T cell response and antitumor efficacy as candidate vaccines. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCC epitopes. Four HCC epitopes MAGE-1(278-286aa), MAGE-3(271-279aa), AFP1 (158-166aa) or AFP2 (542-550aa) were fused to the 3' terminus of the truncated HBV core gene, respectively, or conjunctively. Not all recombinant plasmids led to expression of chimeric proteins in expression strain E. coli BL21 (DE3), but chimeric proteins which are expressed in inclusion bodies resulted in the formation of complete "mature" VLPs. E. coli-derived truncated HBc(1-144) chimeric protein self-assembled into VLPs that both morphologically and physically are similar to the wild-type ones and they still remained activity after purification and refolding from 6M urea solution. We also showed that they could be internalized and presented by DCs in vitro. Additionally, DCs pulsed with the chimeric HBc-VLPs could induce stronger CTL activity and greater IFN-gamma secretion by responding T cells compared with peptid-pulsed DCs. In the B16-pIR-HH tumor therapy model, the growth of established tumors was significantly inhibited by immunization using VLP-pulsed DCs, resulting in significantly higher survival rate of immunized animals. Thus, the results of the current study have demonstrated the principal possibility of using VLP on the basis of HBcAg for creation of a new type of HCC-specific immunogen.

Impaired dendritic cell function resulting from chronic undernutrition disrupts the antigen-specific immune response in mice.

We examined whether antigen-specific immune responses are lower in mice with protein energy malnutrition (PEM mice) compared with nourished (control) mice. The mechanisms underlying reduced antigen-specific immune responses of PEM mice were evaluated through analysis of the functional capacities of antigen-presenting dendritic cells (DC). PEM mice were produced by subjecting male C57BL/6 mice for 52 wk to a daily food intake equivalent to 70% of the mean amount consumed by the control mice that consumed food ad libitum. PEM mice and control mice were immunized with hepatitis B vaccine containing hepatitis B surface antigen (HBsAg) at 52 wk and humoral and cellular immune responses to HBsAg were evaluated at 58 wk. Lymphoproliferative assays were performed to assess the functional capacities of lymphocytes and DC. After 52 wk of food restriction, PEM mice had a 49% lower body weight than controls, almost no subcutaneous fat, severe muscle wasting, and atrophied spleen. All control mice developed antibodies to HBsAg (anti-HBs) in the sera and HBsAg-specific lymphocytes in the spleen as a result of immunization with the hepatitis B vaccine. PEM mice, however, were almost unresponsive to immunization with the hepatitis B vaccine. In PEM mice, the numbers of spleen DC, the T lymphocyte stimulatory capacities of DC, and their production of IL-12p70 and IFN-gamma was less than those of control mice (P < 0.05). We suggest that chronic undernutrition disrupts antigen-specific immune responses and that this disruption can be attributed at least in part to reduced frequencies and impaired functions of DC.

Immunogenicity and safety of an experimental adjuvanted hepatitis B candidate vaccine in liver transplant patients.

Patients with chronic liver disease are at higher risk of hepatitis B (HB) virus infection before and after liver transplantation, and they commonly have a suboptimal immune response to HB vaccines. In this randomized trial, we compared the immunogenicity of primary vaccination with 2 doses of an experimental adjuvanted HB vaccine (adjuvant system 04 containing aluminium and monophosphoryl lipid A [HB-AS04]) to that of 3 double doses of a licensed HB vaccine in 93 liver transplant candidates. Depending on the waiting list for liver transplantation, a booster dose of HB-AS04 or double booster dose of the licensed HB vaccine was given before or after surgery, at 6 to 12 months after initiation of the vaccination course. The percentage of subjects with seroprotective anti-HB surface antibody concentrations 1 month after booster was twice as high in the HB-AS04 group (60.0%), vs. patients in the comparator group (32.0%) (P = 0.035). In subjects who did not undergo liver transplantation before administration of the booster, better immunogenicity results were obtained: 80% of subjects were seroprotected after HB-AS04 vaccination vs. 60% with the comparator (P = 0.2302). Despite a slightly higher reactogenicity, the safety profile of the HB-AS04 vaccine was clinically acceptable. In conclusion, an improved antibody response was observed in liver transplant candidates with 3 doses of HB-AS04, as compared to 4 double doses of a comparator. Liver transplant candidates could benefit from the use of this experimental adjuvanted HB vaccine to further increase their protection against HB infection.