Quantitation of hepatitis B surface antibody by an automated microparticle enzyme immunoassay

A fully automated microparticle enzyme immunoassay, IMx AUSAB, was developed for the detection and quantitation of antibody against hepatitis B surface antigen (anti-HBs). The IMx AUSAB assay can complete 24 tests in less than 45 minutes. Anti-HBs concentrations in specimens are calculated automatically by comparison of the specimen rate to values determined from a stored standard curve. IMx AUSAB sensitivity is 2-3 mIU/ml, equivalent in sensitivity to AUSAB RIA or EIA. Specimens from blood donors, diagnostic and hospital patients, hepatitis B vaccinees, and individuals with a variety of infectious and autoimmune diseases tested in parallel by IMx AUSAB and AUSAB RIA or IMx AUSAB and EIA gave overall qualitative agreement of 97.8% (1265/1293) and 99.1% (1281/1293), respectively. The prevalence of anti-HBs ranged from 5.9% in volunteer blood donors to 47.0% of specimens from a sexually transmitted disease clinic. Most discordant specimens (18/34) were low level reactive (less than 10 mIU/ml) by AUSAB RIA, but negative by IMx AUSAB and AUSAB EIA. These specimens were also negative for antibodies to hepatitis B core antigen (anti-HBc). Six discordants were low level reactive by IMx but negative by RIA and EIA. Three of these six specimens were also reactive for anti-HBc suggesting that the IMx AUSAB reactivity resulted from the presence of low level anti-HBs. Quantitative agreement between IMx AUSAB and RIA or IMx and EIA for 106 specimens ranging in anti-HBs concentration from 1 to 30,000 mIU/ml gave linear correlation coefficients of 0.91 and 0.96, respectively. The IMx test was useful for monitoring hepatitis B vaccine response and seroconversion levels after hepatitis B infection.     

Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg

A second generation radioimmunoassay (RIA) and enzyme-linked immunoassay (EIA) for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) was developed which utilizes recombinant DNA-derived HBsAg (rHBsAg) in place of human plasma derived HBsAg. In these sandwich assays, rHBsAg immobilized on a solid phase was used to capture anti-HBs from the specimen and rHBsAg conjugated to horseradish peroxidase or radiolabeled with 125I was used as a detecting reagent. These rHBsAg-based assays were compared to a commercial radioimmunoassay for anti-HBs detection (AUSAB RIA). For a population of 1711 sera and plasma specimens, 99.2% overall agreement was demonstrated between the recombinant RIA and EIA and 98.6% agreement was observed between the recombinant assays and AUSAB-RIA. The recombinant assays demonstrated equivalent sensitivity and detectability to AUSAB RIA. Most discrepant samples were low-level reactive by AUSAB-RIA, generally less than 10 mIU/ml, and likely represent nonspecific reactivity since no other marker for hepatitis B infection was detected in these samples.     

Comparison of two techniques for detecting antibody to HBsAg during a hepatitis B vaccine study

Two assays for the detection of antibody against hepatitis B surface antigen (anti-HBs) were compared. The first was a direct sandwich radioimmunoassay (RIA) which detects, in principle, antibody against any epitope of hepatitis B surface antigen (HBsAg). The second assay was an inhibition enzyme-linked immunosorbent assay (ELISA). In this assay a fixed amount of HBsAg which can be blocked by anti-HBs is measured in a direct sandwich test. Prevaccination screening sera (n = 191) and follow-up sera obtained from high risk groups (n₁ = 85; n₂ = 41) during two hepatitis B vaccine studies were compared in RIA and ELISA. In prevaccination sera either HBsAg or anti-HBs were detected by ELISA. Full agreement between the results of RIA and ELISA for anti-HBs was obtained in sera containing more than 10 IU/1 anti-HBs. Both tests showed variable results at low titres. Experiments with monoclonal anti-HBs indicated that ELISA is less sensitive for subtype specific antibodies (anti-d, anti-y), which may explain that there were consistent differences between RIA and ELISA in a minority of cases.     

Highly Sensitive Detection of Hepatitis B Virus Surface Antigen by Use of a Semiautomated Immune Complex Transfer Chemiluminescence Enzyme Immunoassay

The performance of hepatitis B surface antigen (HBsAg) screening assays is continuously improved to reduce the risk of transfusion-associated hepatitis B. In this study, a semiautomated immune complex transfer chemiluminescence enzyme immunoassay (ICT-CLEIA) for the detection of HBsAg, which is as sensitive as hepatitis B virus (HBV) DNA PCR, was developed; the ICT-CLEIA assay performance was compared with the performance of the Architect HBsAg QT assay and HBV DNA PCR. The specificities in the initial assay and after retesting were 99.50% (1,988/1,998 samples) and 99.95% (1,997/1,998 samples), respectively. The analytical detection limit was determined to be 0.2 mIU/ml using the 2nd International WHO HBsAg standard, and the cutoff value (0.5 mIU/ml) of the ICT-CLEIA assay was 8.0 standard deviations (SD) above the mean of the HBsAg-negative specimens. The ICT-CLEIA assay could detect HBsAg even in the presence of anti-HBs antibodies and demonstrated a 23.6-day-shorter window period using commercially available HBsAg seroconversion panels than the Architect HBsAg QT assay. Furthermore, the monitoring of the viral kinetics by the ICT-CLEIA assay and the HBV DNA PCR produced very similarly shaped curves during both the HBsAg seroconversion and reverse seroconversion periods. Therefore, the ICT-CLEIA assay may be useful not only for an earlier detection of HBV reactivation but also for the monitoring of hepatitis B patients.     

不同剂量乙肝疫苗与HBIG联合免疫阻断HBV母婴传播的效果对比

目的 探讨10 μg和20 μg乙肝疫苗与HBIG联合免疫阻断HBV母婴传播的效果.方法 124例HBsAg阳性孕妇所生的婴儿随机分为两组,即10 μg乙肝疫苗组和20 μg乙肝疫苗组.婴儿于出生6h内及30 d分别注射200 IU HBIG,同时分别于出生24 h内、1个月及6个月注射3次10 μg或20 μg重组酵母乙肝疫苗.检测婴儿出生时以及1岁时血清HBV标志物.结果 两组新生儿血清HBsAg、HBeAg及抗-HBe阳性率与滴度之间差别均无统计学意义（P＞0.05）.所有新生儿血清HBV DNA水平均小于检测下限（500 U/ml）.出生12个月时,所有124例婴儿血清HBsAg和HBeAg检测结果均为阴性;血清HBV DNA水平均在检测下限以下;10 μg和20 μg乙肝疫苗组血清抗-HBs阳性率分别为90.3％和96.8％,差异无统计学意义（P＞0.05）;抗-HBs水平分别为325.5±342.2 mIU/ml和463.7±353.3 mIU/ml,后者显著高于前者（P=0.01）.而且,20 μg乙肝疫苗组产生高应答抗-HBs（＞ 100 mIU/ml）的比例显著高于10μg乙肝疫苗组（P =0.035）.结论 20 μg乙肝疫苗联合HBIG方案阻断HBV母婴传播的效果优于10 μg乙肝疫苗联合HBIG方案.     

Modellversuche zur Entwicklung einer Hepatitis B Vaccine: ImmunogenitÄt von HBsAg im Meerschweinchen

Zusammenfassung Hepatitis B surface Antigen (HBsAg) wurde aus menschlichem Plasma durch Gelchromatographie, isopkynische Zentrifugation und zonale Sedimentation weitgehend von Plasmaproteinen und Hepatitis B Viruspartikeln (HBV) mit 60%iger AktivitÄtserhaltung gereinigt. Eine Behandlung mit Formaldehydkonzentrationen bis 0,1% zur Inaktivierung möglicher RestinfektiositÄt im gereinigten HBsAg reduziert die AntigenitÄt in vitro und die ImmunogenitÄt im Meerschweinchen nur gering. Adsorption an Aluminiumhydroxyd ergibt etwa 16 mal höhere Werte an Antikörpern gegen HBsAg (anti-HBs) als die Verabreichung von gelöstem HBsAg. Nach zwei Injektionen von 0,2g Formaldehyd-behandeltem HBsAg mit Aluminiumhydroxyd wurde in Meerschweinchen ein mittlerer anti-HBs-Titer von 4 internationalen Einheiten/ml (Normalwert bei menschlichen Rekonvaleszenten etwa 0,1) gefunden, der ohne weitere Injektionen für ein Jahr stabil blieb. Eine passive Verabreichung von einem zwölffachen Äquivalent an Meerschweinchen-anti-HBs kurz vor der ersten Injektion von HBsAg beeintrÄchtigte die aktive Bildung von anti-HBs nach einer zweiten Injektion nicht. Zur Verringerung eines nicht völlig auszuschlie\enden Infektionsrisikos und zur Vermittlung eines raschen Schutzes könnte bei zukünftigen Versuchen mit einer HBsAg-Vaccine gleichzeitig Hepatitis B Immunoglobulin verabreicht werden. Um eine möglichst vollstÄndige Freiheit von HBV in der Vaccine zu gewÄhrleisten, sollte neben einer effizienten Reinigung und Inaktivierung als Ausgangsmaterial anti-HBe-positives Plasma verwendet werden.

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Experiments for the development of a hepatitis B vaccine: Immunogenicity of HBsAg in guinea pigs

Hepatitis B surface antigen (HBsAg) was purified from human plasma by gel chromatography, isopyknic centrifugation, and zonal centrifugation. The final product had about 60% of the original activity and was essentially free from hepatitis B virus particles (HBV) and plasma proteins. Treatment with formaldehyde concentrations up to 0.1% for inactivation of residual infectivity did not significantly reduce antigenicity in vitro and immunogenicity in guinea pigs. Adsorption to aluminum hydroxide resulted in 16-fold higher concentrations of antibody against HBsAg (anti-HBs) than did injection of soluble HBsAg. After two injections of 0.2g HBsAg, which was treated with 0.1% formaldehyde and absorbed to aluminum hydroxide, the median titer of anti-HBs in guinea pigs was 4 IU/ml (normal value in human hepatitis B convalescents: about 0.1) for 1 year without further injections. When guinea pigs received 12 equivalents of homologous anti-HBs serum before the first injection of adsorbed HBsAg, the same anti-HBs titers were found after the booster injection as in animals which had not been passively immunized. A simultaneous application of an experimental HBsAg vaccine and hepatitis B immunoglobulin would probably decrease the potential risk of HBV infections caused by the vaccine itself and also produce rapid protection. To establish absence of HBV as completely as possible, the vaccine should be produced from anti-HBe-positive plasma by efficient purification procedures and it should be inactivated by formalin.

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Mit Unterstützung des Bundesgesundheitsministeriums für Jugend, Familie und Gesundheit und der Deutschen Forschungsgemeinschaft     

Long-term follow-up of Hepatitis B Surface antibody levels in subjects receiving universal Hepatitis B vaccination in infancy in an area of hyperendemicity: correlation between radioimmunoassay and enzyme immunoassay

The aims of the present study were to determine (i) the long-term immunogenicity and the decay rate of hepatitis B virus (HBV) surface antibody (anti-HBs) from universal hepatitis B vaccination at infancy for a healthy population in an area of hyperendemicity and (ii) whether the anti-HBs levels measured by enzyme immunoassay (EIA) were closely correlated with those assayed by radioimmunoassay (RIA) methods during long-term monitoring. A total of 1,337 apparently healthy children (696 boys and 641 girls) who were vaccinated against HBV at infancy and monitored for anti-HBs annually from 7 to 16 years of age entered the study. Serum samples were analyzed for anti-HBs by RIA at 7 to 15 years of age and were also analyzed by EIA at 13 to 16 years of age. Antibody titers were quantified in mIU/ml by EIA as well as by the ratio of the count in the sample to the count for a negative control (S/N) by RIA. In non-boosted children, the average decay of anti-HBs from 7 to 16 years of ages indicated that approximately 20% of the geometric mean titer decays per year. There was a good correlation between serum anti-HBs levels measured by the RIA and the EIA methods (r=0.91; P<0.0001). An equation for RIA to EIA level conversion was established: log EIA titer=-0.12+ (1.31 . log RIA S/N). The anti-HBs titers measured by EIA correlate well with the S/N assayed by RIA. The annual decay rate of the log anti-HBs level may help in planning booster immunizations for hypo-responders or individuals at risk in adolescence.     

Hepatitis B immunization in high risk neonates born from HBsAg positive mothers: comparison between plasma derived and recombinant DNA vaccine

A half dose recombinant hepatitis B vaccine (HBVax II, MSD, 5 micrograms) was investigated for efficacy in the prevention of perinatal hepatitis B virus (HBV) transmission in high risk neonates born from e-antigen positive HBsAg carrier mothers as compared to the half-standard dose regimen of plasma derived hepatitis B vaccine (HBVax, MSD, 10 micrograms). Forty infants born to carrier mothers were given hepatitis B immune globulin (HBIG) 100 IU intramuscularly immediately after birth, combined with either the recombinant or plasma derived hepatitis B vaccine. The infants were randomly divided into two groups of 20 infants each. The plasma derived vaccine (10 micrograms) was given to group I, while infants in group II received the recombinant vaccine (5 micrograms) at birth, 1 and 6 months of age. There were no statistically significant differences in the efficacy and the seroconversion rate of these two combined prophylaxis regimens. The protective efficacy rate of both kinds of HBV vaccine was found to be 94.6 and 89.2 percent in group I and group II respectively. At twelve months of age, the anti-HBs seroconversion rates were 95.0 percent in group I and 84.2 percent in group II. However, the geometric mean titres in group I (179.55 mIU/ml) was significantly higher than those in group II (42.2 mIU/ml) but the anti-HBs titre was still above protective level (10 mIU/ml) in most of the infants.(ABSTRACT TRUNCATED AT 250 WORDS)     

The utilization of biotin-antibiotin interaction for the detection of antibody to hepatitis B surface antigen (anti-HBs)

A biotin-antibiotin solid-phase enzyme-linked immunoassay for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) is described. The assay utilizes hepatitis B surface antigen as a solid-phase 'capture' reagent and a mixture of biotinylated HBsAg and antibiotin-conjugated horseradish peroxidase as a detector reagent. The assay was compared to a commercial enzyme immunoassay (AUSAB EIA) which used the biotin-avidin system for anti-HBs detection. The two assays were found to measure the same molecules and to correlate well regarding anti-HBs titers.     

Long-Term Follow-Up of Hepatitis B Surface Antibody Levels in Subjects Receiving Universal Hepatitis B Vaccination in Infancy in an Area of Hyperendemicity: Correlation between Radioimmunoassay and Enzyme Immunoassay

The aims of the present study were to determine (i) the long-term immunogenicity and the decay rate of hepatitis B virus (HBV) surface antibody (anti-HBs) from universal hepatitis B vaccination at infancy for a healthy population in an area of hyperendemicity and (ii) whether the anti-HBs levels measured by enzyme immunoassay (EIA) were closely correlated with those assayed by radioimmunoassay (RIA) methods during long-term monitoring. A total of 1,337 apparently healthy children (696 boys and 641 girls) who were vaccinated against HBV at infancy and monitored for anti-HBs annually from 7 to 16 years of age entered the study. Serum samples were analyzed for anti-HBs by RIA at 7 to 15 years of age and were also analyzed by EIA at 13 to 16 years of age. Antibody titers were quantified in mIU/ml by EIA as well as by the ratio of the count in the sample to the count for a negative control (S/N) by RIA. In nonboosted children, the average decay of anti-HBs from 7 to 16 years of ages indicated that approximately 20% of the geometric mean titer decays per year. There was a good correlation between serum anti-HBs levels measured by the RIA and the EIA methods (r = 0.91; P < 0.0001). An equation for RIA to EIA level conversion was established: log EIA titer = −0.12 + (1.31 · log RIA S/N). The anti-HBs titers measured by EIA correlate well with the S/N assayed by RIA. The annual decay rate of the log anti-HBs level may help in planning booster immunizations for hyporesponders or individuals at risk in adolescence.     

Vaccination against hepatitis B virus at the Lyon Pasteur Institute. A seven-year evaluation]

Results of immunization against hepatitis B among Pasteur Institute staff members are reported. Prior to immunization, 439 subjects were tested for hepatitis B virus (HBV) markers, including HBs antigen, anti-HBs antibody, and anti-HBc antibody (Ausria, Ausab, Corab assays; Abbott). Forty-seven subjects tested positive for anti-HBs antibody. 317 subjects negative for all the HBs markers studied were given three intramuscular doses of Hevac B (Pasteur vaccins) at one-month intervals. Anti-HBs antibodies were assayed after the third injection with the following results: mean titer, 1,454 mIU/ml, standard deviation, 5,349 mIU/ml, and range, 4 to 41,100 mIU/ml. Anti-HBs titers above 10 mIU/ml were found in 879.4% of subjects. Non-responders and weak responders (anti-HBs titer under 10 mIU/ml) were given a fourth dose of vaccine. Ultimately, after the last (third of fourth) injection 97.6% of subjects had protective antibody titers. No case of HBV infection was seen during the seven-year follow-up period.     

Hepatitis B virus (HBV) infections in turtles

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.     

Application of a Newly Developed High-Sensitivity HBsAg Chemiluminescent Enzyme Immunoassay for Hepatitis B Patients with HBsAg Seroclearance

We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring.     

Antibodies against Hepatitis A and Hepatitis B Virus in Intravenous Immunoglobulin Products

The worldwide seroprevalence of hepatitis A virus (HAV) and hepatitis B virus (HBV) has changed over the last two decades, indicating a declining incidence of HAV and HBV infections. Therefore, vaccinations against HAV and HBV are recommended for unimmunized people before traveling to an endemic area. Unfortunately, primary antibody deficiency (PAD) patients can only obtain humoral immunity through intravenous immunoglobulin G (IVIG) replacement and not from vaccination because of a defect in antibody production. However, few studies have analyzed the titers of antibodies against HAV or HBV in IVIG products. In this study, the titers of anti-HAV and anti-HBs antibodies were measured in nineteen lots of IVIG products from five manufacturers from three countries (A, B from Korea; C, D from Japan; and E from the USA), and trough titers in plasma were estimated. Concentrations of anti-HAV antibody ranged from 1,888–8,927 mIU/mL and estimated trough titers exceeded the minimal protective value in all evaluated IVIG products. Concentrations of anti-HBs antibody ranged from 438–965 mIU/mL in products A and B and were 157, 123, and 1,945 mIU/mL in products C, D, and E, respectively. Estimated trough titers in products A, B, and E exceeded the minimal protective value but those in products C and D did not reach this threshold. These data demonstrated that available IVIG products generally provide sufficient antibodies against HAV and HBV to protect patients with PAD, although the trough concentrations of anti-HBs antibody in two IVIG products did not reach the minimum protective value.     

Long-term anti-HBs antibody persistence and immune memory in children and adolescents who received routine childhood hepatitis B vaccination

This paper presents data from two studies that evaluated 5-y and 10-y persistence of antibodies against hepatitis B (HBV) surface antigen (anti-HBs) and immune response to an HBV vaccine challenge in children and adolescents who had received three doses of a HBV vaccine in infancy as part of routine clinical practice [NCT00519649/NCT00984139]. Anti-HBs antibody concentrations ≥ 10 mIU/ml persisted in 83.3% (95% confidence interval [CI]: 78.5–87.5) and 78.3% (95% CI: 73.1–83.0) of subjects aged 7–8 y and 12–13 y, respectively 5–10 y after infant vaccination. One month postchallenge dose, 98.2% (95% CI: 95.9–99.4) and 93.7% (95% CI: 90.2–96.2) of subjects in the two age groups, respectively had anti-HBs antibody concentrations ≥ 100 mIU/ml. Overall, 99.6% (95% CI: 98–100) and 97.2% (95% CI: 94.5–98.8) of subjects aged 7–8 y and 12–13 y mounted an anamnestic response to the HBV challenge dose, which was well-tolerated. Healthy children aged 7–8 y and adolescents aged 12–13 y received three doses of a monovalent pediatric HBV vaccine (10 μg of HBsAg) before 18 mo of age. Serum samples collected before and one month post-HBV vaccine challenge dose were tested for anti-HBs antibody concentrations. Safety assessments were made for the HBV vaccine challenge dose. A three-dose childhood HBV immunization regimen induced persistence of antibodies against HBV infection for 10 y, up to adolescence. This vaccination regimen also conferred long-term immune memory against HBV as evidenced by the strong anamnestic response to the HBV vaccine challenge, despite waning anti-HBs antibody levels.     

Cell-mediated immunity to hepatitis B surface antigen in man.

An aqueous preparation of hepatitis B virus (HBV) vaccine was used as an intradermal skin test antigen to assess delayed hypersensitivity to hepatitis B surface antigen (HBsAg). Thirty-five persons were tested including 10 individuals seronegative for all HBV markers, 10 positive for HBsAg (chronic carriers) and 15 positive for antibody to HBsAg (anti-HBs), five of whom had received the HBV vaccine. All patients were also studied for lymphocyte blastogenic responses to phytohaemagglutinin, concanavalin A, pokeweed mitogen and purified HBsAg. Only one individual had a positive delayed skin test reaction to HBsAg. This person had received the HBV vaccine and had high titres of anti-HBs in serum. However, neither this individual nor any other subject exhibited a positive lymphocyte blastogenic response to HBsAg in vitro. Thus, delayed hypersensitivity skin test reactivity to HBsAg was not detected after natural infection with HBV and was rarely present in hyperimmunized individuals. In vitro assays of immune responsiveness failed to demonstrate cellular immunity to HBsAg even in hyperimmunized persons. These studies provide no evidence that cell-mediated immunity to HBsAg plays a role in the immunopathogenesis of acute or chronic type B hepatitis.     

Requirement of standardizing anti-HBs assay methods in Japan for HBV infection-preventing strategy--discrepancy of anti-HBs measurements among three different kits widely used in Japan

The strategy to eliminate hepatitis B virus (HBV) infection by administrating an HB vaccine is changing worldwide; however, this is not the case in Japan. An important concern about the HBV infection-preventing strategy in Japan may be that the assay methods for the antibody to hepatitis B surface antigen (anti-HBs) are not standardized. The minimum protective anti-HBs titer against HBV infection has been established as 10 mIU/ml by World Health Organization (WHO) -standardized assay methods worldwide, but that is still determined as a "positive" test result by the passive hemagglutination (PHA) method in Japan. We compared anti-HBs measurements in given samples among PHA(Mycell II, Institute of Immunology), chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse, Fujirebio), and chemiluminescent immunoassay (CLIA) (Architect, Abbott), all of which are currently in wide use in Japan. First, anti-HBs measurements in serum from individuals who received a yeast-derived recombinant HB vaccine composed of the major surface protein of either subtype adr or subtype ayw were compared. The results clearly showed that in subtype adr-vaccinees CLIA underestimated the anti-HBs amount compared with CLEIA and PHA, but in ayw-vaccinees, the discordance in the measurements among the three kits was not prominent. Second, anti-HBs measurements in standard or calibration solutions of each assay kit were compared. Surprisingly, CLEIA showed higher measurements in all three kit-associated standard or calibration solutions than CLIA. Thus, the anti-HBs titer of 10 mIU/ml is difficult to introduce in Japan as the minimum protective level against HBV infection. Efforts to standardize anti-HBs assay methods are expected to share international evidence about the HBV infection-preventing strategy.     

Low-dose vaccination against hepatitis B in children: one-year follow-up

Six hundred forty-three children, negative for markers of hepatitis B virus (HBV) infections, were given three X 2-micrograms doses of Merck, Sharp and Dohme (MSD) plasma derived hepatitis B vaccine (H-B-Vax) at monthly intervals. Twelve months after the first dose of vaccine, antibody to hepatitis B surface antigen (anti-HBs) was detected in 89% of children by radioimmunoassay (RIA) and in 83% by enzyme immunoassay (EIA). Seroconversion rates and anti-HBs titres were significantly greater in 1-4-year-olds than in older children (p less than 0.01). Eighteen children with no anti-HBs or other markers of HBV at this time were given 10 micrograms of vaccine and tested one month later. Seventeen developed anti-HBs, 12 at levels consistent with an anamnestic response. Forty-nine HBV-marker-negative children seroconverted for antibody to hepatitis B core antigen (anti-HBc) in the 8-month period before or the 12-month period following vaccination. Forty-six of these children were positive for anti-HBs, and one has been confirmed as a chronic carrier of hepatitis B surface antigen (HBsAg). Three cases of clinical hepatitis B in children have been seen in the community since the vaccination programme began. Two of these were amongst the estimated 5% of children who were not vaccinated. The third was in a vaccinee and occurred 4 1/2 months after the last dose of vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute hepatitis B viral infection in a patient with anti-HBs.

We report a patient with antibody to hepatitis B surface antigen (anti-HBs) but no antibodies to other hepatitis B virus components, who developed acute symptomatic type B hepatitis. The possible explanations for this unusual serological pattern are 1) the antibody-positive status, which developed against only a subdeterminant of hepatitis B surface antigen (HBsAg), arose naturally or as the result of cross-reaction with a variety of antigens; and 2) seroconversion to anti-HBs occurred in response to surface antigen of a mutant strain of hepatitis B virus (HBV). This anti-HBs positivity, in the absence of antibody to hepatitis B core antigen, does not provide natural immunization against HBV infection, and so is not protective. Individuals who are positive to anti-HBs antibody alone which is not elicited by HBV vaccine, should be vaccinated against possible HBV infection.     

Production and efficacy of a dendritic cell-based therapeutic vaccine for murine chronic hepatitis B virus carrierer

Recently a new field of immunological research and clinical application of vaccines for therapeutic purposes (vaccine therapy) has been developed for treating several chronic viral infections including chronic hepatitis B virus (HBV) infection. Administration of vaccine containing hepatitis B surface antigen (HBsAg) for 1 year has resulted in negative HBsAg and development of antibody to HBsAg (anti-HBs) in some, but not in all, HBV transgenic mouse (HBV-Tg). In order to develop more potent regimen of vaccine therapy for chronic HBV carrier, we prepared a dendritic cell (DC)-based therapeutic vaccine and evaluated their therapeutic potential in HBV-Tg. DCs were isolated from single cell suspensions of murine spleen cells by collagenase digestion, density centrifugation and depletion of lymphocytes. Spleen DCs were cultured with HBsAg (100 microg) for 24 h to produce HBsAg-pulsed DCs. HBV-Tg expressing HBsAg and HBV DNA in the sera were randomly assigned to receive either HBsAg-pulsed DCs (n = 20) or unpulsed DC (n = 20) or vaccine containing HBsAg (n = 39) or complete Freund's adjuvant (n = 20) or left untreated (n = 20). Only two intraperitoneal injections of HBsAg-pulsed DCs resulted in negative HBsAg and production of anti-HBs in the sera in all HBV-Tg (n = 20). However, administration of un-pulsed DCs (n = 20) or vaccine containing HBsAg (n = 39) or only complete Freund's adjuvant did not induce negative HBsAg or production of anti-HBs in any HBV-Tg within 6 months of therapy commencement. Taken together, this study showed that HBsAg-pulsed DCs represent a highly potent therapeutic vaccine for chronic HBV infection and inspire optimism of using this vaccine in clinical conditions. A clinical trial of HBsAg-pulsed DC in patients with chronic hepatitis B is warranted.