Heijtink, R.A.

Genetic characteristics of hepatitis B virus genotypes as a factor for interferon-induced HBeAg clearance (Article)

2007-08-01T00:00:00Z

The factors determining the responsiveness of different hepatitis B virus (HBV) genotypes to interferon treatment are not fully understood. We investigated the relationship between HBV genetic characteristics and the outcome of short (16 weeks) or prolonged (32 weeks) treatment with standard interferon-alpha in a prospectively followed cohort of 103 patients across Europe with HBeAg positive chronic hepatitis B. INNO-LiPA assays and HBV DNA sequencing were used to determine HBV genotypes, mutations in the core promoter and precore/core regions. After 16-weeks interferon-alpha treatment, the rate of HBeAg clearance was higher in genotype A versus all other genotypes (P = 0.014), or genotype D alone (P = 0.05). The HBV genome analysis revealed that: (i) after 16-weeks treatment, an HBV subpopulation with core promoter mutations emerged or increased (P < 0.001) only in genotype A; (ii) the core gene of genotype A has the lowest number of amino acid variations in comparison with genotypes B, C, or D. Logistic regression analysis identified genotype A as a positive predictor of short (16 weeks) treatment response (P = 0.001; odds ratio 6.19, 95 confidence interval 1.94-19.8), having a greater impact than baseline HBV DNA or alanine aminotransferase (ALT) levels. In contrast, the response to prolonged interferon-alpha treatment was not different between HBV genotypes. These results suggest that HBV genotype A responds earlier to interferon treatment than other genotypes, which is associated with its molecular characteristics. The optimal duration of interferon-based therapies in chronic hepatitis B may vary between different HBV genotypes.

Hepatitis B surface antigen (HBsAg) derived from yeast cells (Hansenula polymorpha) used to establish an influence of antigenic subtype (adw2, adr, ayw3) in measuring the immune response after vaccination. (Article)

2002-05-22T00:00:00Z

As a product of western world biotechnology the yeast (Saccharomyces cerevisiae) hepatitis B vaccine was introduced as antigenic subtype adw2. However, an HBsAg/adw2-vaccine may provide a good but not "optimal" immunologic response for infection with heterologous virus strains. The availability of the yeast Hansenula polymorpha HBsAg in three different antigenic forms (adw2, ayw3 and adr) enabled us to investigate the influence of variant amino acids in the binding of immune anti-HBs after vaccination. Hansenula-derived HBsAg was standardised on the basis of protein content at >95% purity. Standardisation was controlled by monoclonal anti-HBs binding in a well-conserved region. Sera were obtained after immunisation with type adw, ayw and adr vaccines. Direct binding of immune antibodies to homologous antigen (in EIA) was higher than to heterologous antigen except for the adr-related antibodies. Since the binding of the WHO reference anti-HBs was strongly reduced for the ayw and adr compared to the adw antigen, a similar binding profile for the three antigens on protein basis could result in 2-3-fold different anti-HBs level expressed in IU/l. Inhibition of Hansenula-derived HBsAg binding to solid phase monoclonal anti-HBs in enzyme immunoassays after incubation with serum anti-HBs confirmed the differential binding of serum anti-HBs with variant Hansenula-derived HBsAg. This variant (antigenic subtype) dependent reactivity of anti-HBs in immunoassays in combination with a variant specific WHO standard may limit the application of the threshold levels of 10 and 100 IU/l for seroconversion and seroprotection.

Characterization of a human monoclonal antibody obtained after immunization with plasma vaccine and a booster with recombinant-DNA hepatitis B vaccine. (Article)

2002-02-11T00:00:00Z

A human monoclonal antibody type IgG4, designated 1Ff4, was obtained by Epstein Barr virus transformation of peripheral blood lymphocytes from a hepatitis B vaccinee (HB-VAX: plasma-derived vaccine) after one boost of yeast recombinant DNA derived vaccine (Engerix-B). 1Ff4 binds preferentially to HBsAg/adw(2) and HBsAg/ayw(1). In binding experiments, it competes with antibodies induced by vaccination with HB-VAX-DNA (yeast recombinant) and HB-VAX (plasma-derived vaccine). 1Ff4 competes in part with a monoclonal antibody for the w/r region. Partial inhibition of binding of HBsAg/adw(2) to solid phase anti-HBs was detected, resembling inhibition obtained using other human monoclonal specific for the "a"-loop. 1Ff4 does not bind to linear peptides covering the two "a"-loops or to an adw(2)/G145R mutant, its binding to wild type HBsAg strongly depends on the presence of disulphide bonds. In a large series of HBsAg-positive samples from an endemic area, 1Ff4 antibodies were successfully used to discriminate between an adw(2) and an adrq+ strain. The characterisation of 1Ff4 and other human monoclonal anti-HBs antibodies may help to understand the fine specificity of protective antibodies elicited by immunization.

Anti-HBs after hepatitis B immunization with plasma-derived and recombinant DNA-derived vaccines: binding to mutant HBsAg. (Article)

2001-06-14T00:00:00Z

The G145R mutant of the small S-protein is a major escape mutant of hepatitis B virus observed in natural infection, after immunization and HBIG therapy. In a previous study we found that plasma-derived and recombinant DNA-derived vaccine HBsAg reacted differently with monoclonal antibodies sensitive for the G145R change. In the present study we investigated the binding of polyclonal anti-HBs obtained after immunization with plasma vaccine and recombinant DNA vaccine to synthetic peptides (adw(2), adr) and rHBsAg (HepG2) (ayw(3); wild type and a 145R mutant). Anti-HBs binding to synthetic peptids (25-mers, 7aa overlap) from the "a"-loop was significantly reduced by the G145R substitution and by changing the amino acid sequence from adw(2) into adr. With mutant G145R rHBsAg the inhibitory activity of vaccine anti-HBs was decreased compared to rHBsAg wild type. In general only minor differences were observed between plasma vaccine and recombinant DNA vaccine related antibody responses. However, the individual heterogeneity in epitope specific reactivity with its possible consequences for protection (against escape mutants) is not reflected in an anti-HBs titer by standard anti-HBs assays. The presented differentiation in anti-HBs response after immunization may deliver new tools for evaluation of future vaccines.

Administration of a human monoclonal antibody (TUVIRUMAB) to chronic hepatitis B patients pre-treated with lamivudine: Monitoring of serum TUVIRUMAB in immune complexes. (Article)

2001-01-01T00:00:00Z

A human monoclonal anti-hepatitis B antibody preparation (TUVIRUMAB) was administered 6 times over a 2-week period in a dose-escalating schemeto chronic hepatitisBpatients pre-treated with lamivudine. The capacity of the TUVIRUMAB antibody to ``neutralize'' hepatitis B surface antigen in the circulation was investigated by means of experimental enzyme-immunoassays. Monoclonal antibody conjugates enabled the detection of HBsAg, TUVIRUMAB, and HBsAg/ TUVIRUMAB complexes. The results showed that (1) TUVIRUMAB was able partially to ``neutralize'' in vitro and in vivo, (2) HBsAg/TUVIRUMAB complexes can be traced by assays that capture the complex at either its HBsAg or its TUVIRUMAB component, (3) the ®nal concentration of TUVIRUMAB at the end of therapy varied greatly but seemed to be related to HBsAg production at the start of therapy, (4) for at least 14 days after discontinuation of therapy, a minimal HBsAg level could be maintained in the presence of a declining TUVIRUMAB titer in patients with less than 3 mg/ml HBsAg before the start of therapy, (5) three months after therapy, all HBsAg levels had returned to pretreatment levels and TUVIRUMAB had disappeared.

Anti-HBs characteristics after hepatitis B immunisation with plasma-derived and recombinant DNA-derived vaccines. (Article)

2000-02-14T00:00:00Z

Hepatitis B surface antigen derived from chronic hepatitis B carriers has been replaced almost completely by recombinant DNA-derived HBsAg for use as hepatitis B vaccine. Similarly, recombinant DNA-derived HBsAg is replacing plasma-derived HBsAg in standard anti-HBs assays. We analysed the influence of a change from plasma-derived HBsAg to recombinant DNA-derived HBsAg on antigen presentation in immunoassays and the characteristics of the anti-HBs antibodies after immunisation. Antigens and/or antibodies were subjected to three types of experiments: (a) binding of ‘a’-loop specific monoclonal (anti-S) antibody conjugates to immobilised vaccine-HBsAg; (b) binding of post-vaccination anti-HBs to immobilised (vaccine-)HBsAg and (c) inhibition of HBsAg binding to immobilised monoclonal anti-HBs after pre-incubation with post-vaccination antibodies. Our results show that, in both antigen presentation and anti-HBs binding properties, yeast recombinant HBsAg and related antibodies could be clearly distinguished from plasma-derived HBsAg and related antibodies. Divergent results were also obtained in the inhibition assay with recombinant DNA-derived HBsAg but not with serum HBsAg from the vaccine HBsAg subtype. It is concluded that both antigen presentation in vaccines and in anti-HBs assays can markedly influence the quantitation anti-HBs response. It is suggested that a challenge with an heterologous hepatitis B virus may encounter reduced efficacy of vaccine antibodies.

Characteristics of early phase of chronicity in acute hepatitis B infection. (Article)

1999-01-01T00:00:00Z

The mechanism of development of chronicity after acute hepatitis B infection has not been elucidated fully. Following a single source outbreak of hepatitis B among 79 adult women, three patients (4%) became chronic carriers of hepatitis B virus (HBV). We compared features of the virus and antibody response of the latter three patients with those of 12 HBeAg-positive cases with resolving infection. The virus genotype was D, antigenic subtype ayw2. Base sequence analysis of S- and C-gene regions revealed no differences between the two groups. During the acute illness the three patients who developed chronicity had a remarkable transient reduction of HBsAg, HBeAg, and HBV DNA levels at 14-20 weeks after infection, the time of HBeAg seroconversion in the patients who cleared the infection. One HBeAg-specific monoclonal antibody (HBOT.95A) used as solid-phase antibody in a sandwich enzyme immunoassay detected an increased HBeAg signal in 2 of the 3 patients that developed chronicity and in 1 of the 12 patients who recovered. The latter patient had an exceptional long period of HBsAg antigenemia. Standard HBeAg assays detected HBeAg in all cases. HBeAg and anti-HBe-positive serum samples from the patients who recovered could inhibit the HBOT.95A response. The results suggest that chronic hepatitis B develops after an interruption of immune clearance. Differentiation of the antibody response to HBeAg may help to find patients with an increased risk for this interrupted immune clearance who might be candidates for an early intervention therapy.

Inhibitory effects of acyclic nucleoside phosphonates on human hepatitis B virus and duck hepatitis B virus infections in tissue culture (Article)

1994-01-01T00:00:00Z

The inhibitory effects of the 9-(2-phosphonylmethoxyethyl)adenine-related compounds (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine, (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine, (R)-9-(2-phosphonylmethoxypropyl)adenine, (R)-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine, and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine on human hepatitis B virus replication in the human hepatoma cell line HepG2 2.2.15 and duck hepatitis B virus infection in primary duck hepatocytes were investigated. (R)-9-(2-phosphonylmethoxypropyl-2,6-diaminopurine had the lowest 50% inhibitory concentrations against hepatitis B virus and duck hepatitis B virus, 0.22 and 0.06 microM, respectively, i.e., two- to fivefold lower concentrations than required for (R)-9-(2-phosphonylmethoxypropyl)adenine and 9-(2-phosphonylmethoxyethyl)adenine. All compounds were not toxic in vitro at a concentration of 100 microM.

Sequence analysis of the 5' untranslated region in isolates of at least four genotypes of hepatitis C virus in The Netherlands (Article)

1994-01-01T00:00:00Z

The RNAs of hepatitis C virus (HCV) isolates from 62 patients with chronic HCV infection were analyzed by direct sequencing of the 5' untranslated region. Two important sequence motifs were recognized: one between positions -170 and -155 and the other between positions -132 and -117. These motifs are partly complementary. All three previously published genotypes were observed; 34 (55%) isolates were classified as type 1 (including prototype [from the United States] and HCV-BK [from Japan] sequences), 11 (18%) were classified as type 2 (including HC-J6 and HC-J8), and 12 (19%) were classified as type 3 (including EB1); one patient was infected with genotypes 1 and 2. Four (6%) isolates showed aberrant sequences and were therefore provisionally classified as genotype 4. These results indicate the significance of sequence variation among the 5' untranslated regions of different HCV genotypes and indicate that this region could possibly be used for consistent genotyping of HCV isolates.