R.A. Heijtink

Ten-year neonatal hepatitis B vaccination program, the Netherlands, 1982–1992: protective efficacy and long-term immunogenicity

From 1982 to 1989, 705 infants born to HBsAg-positive mothers entered the Dutch neonatal hepatitis B vaccination program and received passive-active hepatitis B immunization in three randomized controlled trials testing variations in time of starting active vaccination, dose and type of vaccine, and number of hepatitis B immunoglobulin (HBIg) injections. A meta-analysis of individual patient data of the three randomized trials was performed to determine which independent host and vaccination related factors influence protective efficacy and long-term immunogenicity, and to assess whether hepatitis B vaccination concomitant with standard DKTP vaccination provides optimal protection. Statistical methodology included multivariate logistic regression analysis. Eight infants (1.1%), all born to HBeAg-positive mothers, became HBsAg carriers within the first year of life. The protective efficacy rate (PER) of passive-active immunization at 12 months follow-up was 92% for the total group of children from 114 HBeAg-positive mothers with no significant differences between children starting active immunization at birth or at 3 months of age, between infants starting at 3 months of age receiving one or two doses of HBIg or between those receiving plasma derived or recombinant vaccine. The only factor that affected the PER significantly was the level of maternal HBV DNA; PER was 100% if maternal HBV DNA was < 150 pg ml-1 and 68% for HBV DNA levels > 150 pg ml-1. After 5 years of follow-up, the group that started active immunization at birth had significantly more infants with loss of seroprotection (anti-HBs levels < 10 IU l-1, 15%) than the corresponding group starting at 3 months of age (anti-HBs < 10 IU l-2, 2%). One of 35 children with loss of seroprotection at 2 years became a HBsAg carrier in the fifth year of follow-up. This meta-analysis shows that the protective efficacy of passive-active hepatitis B vaccination is mainly influenced by material HBV DNA levels, and independent of the time of starting active vaccination at birth or at 3 months of age; long-term immunity was enhanced by starting active vaccination concomitant with DKTP vaccination. These findings allow incorporation of hepatitis B vaccine into the standard infant immunization programs for countries with a passive-active immunization strategy for the control of hepatitis B. Additional measures are needed to protect neonates of highly viremic women.

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, i.e., human hepatoma cell lines HepG2 2.2.15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2.15 cells and HB611 cells at a 50% inhibitory concentration (IC50) of 0.7 and 1.2 microM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC50 of 0.2 microM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2.15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3H-methyl]deoxythymidine incorporation was 150 microM for the two human hepatoma cell lines and 40 microM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.

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

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.

Immunogenicity of standard and low dose vaccination using yeast-derived recombinant hepatitis B surface antigen in elderly volunteers

There is no conclusive evidence that age influences the response to vaccination against hepatitis B virus. We therefore studied the immunogenicity of yeast-derived rHBsAg vaccine in elderly volunteers. The study was conducted in the outpatient clinics of an academic and a regional hospital, in a rural family practice and in an urban community centre. We recruited 112 healthy volunteers aged 59 years and over, to whom 10 or 20 micrograms yeast-derived HBsAg was given at 0, 1 and 6 months. Anti-HBs titres were measured by radioimmunoassay at 2, 6 and 7 months. Responders and non-responders were compared using univariate non-parametric tests and multivariate logistic regression analysis. Of the 116 subjects who volunteered to take part in the study, 106 vaccinees completed it. The percentage of subjects with an anti-HBs titre > or = 10 IU l-1 at 7 months was 60% (95% confidence interval: 51-70%; geometric mean titre; 253 IU l-1). Of the factors studied, i.e. setting, age, sex, alcohol consumption, current medication and vaccine dose, the use of medication at the time of the first vaccination was the only independent factor related to the response to vaccination, with a response rate of 78% (95% confidence interval: 66-89%) in those without medication. In elderly subjects, the proportion with protective concentrations of anti-HBs after vaccination with 10 or 20 micrograms yeast-derived recombinant HBsAg in a standard scheme is lower than in healthy adolescents. Within the older age group studied here, the use of medication, probably reflecting general health, is the only significant factor influencing the response to vaccination.

Improvement of hepatitis B-associated glomerulonephritis after antiviral combination therapy

A 9-year-old boy with hepatitis B-associated glomerulonephritis and nephrotic syndrome underwent antiviral combination therapy including interferon and acyclovir. Pretreatment evaluation showed that active hepatitis B virus replication with HBsAg, HBeAg, HBV-DNA and DNA-polymerase had occurred for a period of at least 4 years. Signs of liver disease were minimal; serum amino transferases were normal and liver histology showed chronic persistent hepatitis with positive HBcAg, HBeAg and HBsAg immunofluorescence. A kidney biopsy revealed membranous glomerulonephritis with deposition of HBcAg, HBeAg, IgG, C3, C1q and, on electron microscopy, virus-like particles. After 8 weeks of therapy, active viral replication ceased, HBe seroconversion occurred and the nephrotic syndrome disappeared. One year after treatment, the boy was asymptomatic. No viral markers could be detected in the kidney, but low-grade membranous glomerulonephritis persisted with deposition of C1q, IgG and C3, but not HBeAg, HBsAg or HBcAg. Liver histology showed a minimal aspecific portal infiltrate with weak membrane-bound HBsAg immunofluorescence; no HBcAg could be detected. For patients with active viral replication and deposition of HBc, HBe immune complexes in the kidney, antiviral therapy can be beneficial, even in the absence of active liver disease.

Anti-HBs levels after hepatitis B immunisation depend on test reagents: routinely determined 10 and 100 IU/l seroprotection levels unreliable

In a large series of post-vaccination samples we compared the result of three commercially available anti-HBs assays (AxSYM, Architect and Access) on the quantitation of anti-HBs after immunisation with Engerix-B (HBsAg/ad) and GenHevacB (HBsAg/ay) vaccine. Two of the assays (AxSYM, Architect: Abbott Laboratories) gave related but not identical results with HBsAg from different sources. The result of the third assay (Access, Beckman Coulter) was related to that of AxSYM and Architect only for GenHevacB anti-HBs but differed for Engerix-B anti-HBs (P<0.001). This vaccine dependent discrepancy was also observed with the Vidas anti-HBs assay (BioMerieux). An external WHO reference panel could harmonise geometric mean anti-HBs levels from the four assays for GenHevacB but not for Engerix-B vaccination sera. We conclude that the individually determined anti-HBs level (IU/l) strongly depend on the test reagents and the vaccine under study.

Immune response after vaccination with recombinant hepatitis B vaccine as compared to that after plasma-derived vaccine

Thirty-one individuals (health care workers) were vaccinated with recombinant hepatitis B vaccine (10 microgram dose) and their immune response (anti-HBs) was compared to that of twenty-five health care workers after vaccination with plasma-derived vaccine (20 microgram dose). Although the seroconversion rate and the percentage of anti-HBs/a antibodies at month 7 were comparable, the geometric mean titre of anti-HBs at month 7 was considerably lower for the recombinant vaccine group (857.4 vs. 6736.5 IU/l). However, vaccinees from the two groups showing seroconversion at month 1 had comparable titres at month 7. Raising the dose of HBsAg in the recombinant vaccine may favourably influence the seroconversion rate at month 1 and thereby the immune response after three injections.

In vivo activity of a mixture of two human monoclonal antibodies (anti-HBs) in a chronic hepatitis B virus carrier chimpanzee

A 35-year-old female hepatitis B virus carrier chimpanzee was infused with one dose of a mixture of human monoclonal antibodies 9H9 and 4-7B (antibodies against hepatitis B virus surface antigen; HBsAg). Blood samples were taken before and up to 3 weeks after infusion. HBsAg and antibodies against HBsAg (anti-HBs) were quantified by radioimmunoassay and enzyme immunoassay. Free anti-HBs was never detected. Thirty min after the start of the infusion the HBsAg level was minimal with maximum loading of the chimpanzee HBsAg with human immunoglobulin. HBsAg complexes could be dissociated by acid treatment. The HBsAg level was completely restored on day 7. Similar results were obtained for the preS1-containing particles that may represent the infectious viral particles in the chimpanzee serum. A mouse monoclonal anti-HBs (HBs.OT40) was found to compete with 9H9 in artificial immune complexes with the pre-treatment HBsAg from the chimpanzee. Used as a conjugate, HBs.OT40 yielded a maximum decrease in the signal in the 30 min sample compared to non-competing anti-HBs conjugates. This indicates binding of HBsAg with 9H9 in the circulation of the chimpanzee. Immune-complexed 4-7B could not be detected by its corresponding 4-7B peptide conjugate, probably due to its low concentration in the complexes. It is concluded that human monoclonal anti-HBs can effectively reduce the level of HBsAg in serum from this chronic carrier. Monoclonals 9H9 and 4-7B may complement each other due to their different mechanisms of inactivation, probably with higher efficiency than that monitored by our HBsAg screening assays.

Survival and complications in a cohort of patients with anti-Delta positive liver disease presenting in a tertiary referral clinic

BACKGROUND/AIMS Our aim was to evaluate the clinical outcome and survival of patients with anti-Delta positive liver disease in The Netherlands. METHODS We evaluated those patients visiting our hospital between 1978 and 1993 with respect to clinical, virological and histological parameters. During the follow-up period the occurrence of complications of the liver disease and survival was determined. Thirty patients with a median age of 34 years (range 21-52) were included. RESULTS During an average follow up of 4.8 years, nine patients died. The overall 5-year survival as estimated by Kaplan-Meyer analysis was 71%, which was comparable to hepatitis B cirrhosis patients. However, in the group without active hepatitis B replication (HBeAg-negative) a clear trend towards a worse survival was identified in Delta cirrhosis patients. Complications and deaths occurred exclusively in the patient group with cirrhotic liver disease. The complications (ascites, elevated bilirubin >34 micro mol/l), variceal bleeding and spontaneous bacterial peritonitis) occurred in 52% of the patients with a follow up of more than 6 months (n=27). Fifty-seven percent of those patients died. In our population anti-Delta positive liver disease affects predominantly young patients and is related to advanced liver disease. CONCLUSIONS In view of the high death rate, liver transplantation should be considered when signs or symptoms of decompensated liver disease occur.

Anti-HBs levels in infants of hepatitis B carrier mothers after delayed active immunization with recombinant vaccine concomitant with DTP-polio vaccine: is there need for a second dose of HBIg?

The need for an additional dose of hepatitis B immune globulin (HBIg) was studied by comparing infants receiving 1 ml HBIg at birth followed by hepatitis B immunization, concomitant with DTP-polio vaccine, at 3, 4, 5 and 11 months (schedule E), with infants receiving the same schedule with additional HBIg at 3 months (schedule F). The immune response to recombinant hepatitis B vaccine (20 micrograms) was evaluated in 195 infants born to HBsAg-positive mothers allocated to groups E and F and compared with historic controls who received plasma vaccine (10 micrograms) according to schedule F. Blood samples were drawn at 0, 3, 4, 6, 11, 12 and 24 months of age. No difference in efficacy between the two schedules was observed; 8 and 6% of infants born to HBeAg-positive HBsAg carrier mothers in groups E and F, respectively, became HBsAg carriers. Passively acquired antibodies at birth remained present for about 5 months in most infants. The seroprotection rates (anti-HBs > or = 10 IU l-1) were over 90% at all time points and similar for groups E and F. The titres of anti-HBs attained during the first 6 months were statistically lower (p < or = 0.02) for group E than for group F but similar thereafter. Anti-HBs titres in infants receiving the recombinant vaccine were significantly lower than in infants receiving the plasma vaccine (p << 0.001). Supplemental doses of HBIg in infants receiving a high dose of HBIg ( > 200 IU) at birth and the first dose of vaccine at the age of 3 months are not advised.