Occurrence of HBs antigen and anti-HBs antibodies in analytical laboratory workers in Warsaw

The study aimed at answering the question whether markers of the viral hepatitis, namely HBs antigen and anti-HBs antibodies, are significantly more frequent in the personnel of the analytical laboratories than in blood donors of the City Blood Donation Centre. Together 1,284 persons employed at 88 analytical laboratories were examined. These persons were divided into the groups according to the occupation, age and duration of the employment. HBs antigen was detected with EIA technique in 13 subjects making 1,025% of all examined individuals whereas anti-HBs antibodies were detected with EIP technique in 20 subjects, i.e. 1,560%. Detectability of HBs antigen and anti-HBS antibodies in blood donors was 0.443% and 0.04% respectively. The obtained results indicate significantly more frequent occurrence of both markers in the employees of the analytical laboratories.     

Biochemical and immunological properties of a viral hybrid particle expressing the Plasmodium vivax merozoite surface protein 1 C-terminal region

BACKGROUND: Mammalian cells expressing the small hepatitis B virus surface protein (HBs) secrete highly immunogenic 20 nm lipoprotein particles. Previous studies demonstrated that the fusion of foreign sequences into certain regions of HBs leads to chimeric particles carrying epitopes for the foreign peptide, as well as for HBs. The present study investigates immunologic and biochemical properties of the fusion of the C-terminal region of the merozoite surface 1 protein of P. vivax, the most widely distributed human malaria parasite, and HBs (PvMSP1(19)-HBs). MATERIALS AND METHODS: COS7 cells were transfected with a plasmid coding for PvMSP1(19)-HBs. The hybrid products were analyzed by density gradient centrifugation and electron microscopy or detected by metabolic labeling and immunoprecipitation with anti-HBs and patient-derived anti-P. vivax serum. Mice were immunized with the vector and the antibody response was checked by ELISA. RESULTS: The fusion PvMSP1(19)-HBs formed particles of 20-45 nm size, which were secreted from COS7 cells. The particles were immunoprecipitable with anti-HBs and serum of different P. vivax-positive individuals. Immunization of mice with the construct as a genetic vaccine showed that antibodies were raised mostly against the PvMSP1(19) domain and recognized the native protein. CONCLUSION: Due to its biochemical and antigenic properties, the hybrid particle will be useful in future vaccine trials against the asexual blood stages of P. vivax as a genetic and/or a proteic subunit candidate.     

Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus

The susceptibility of the tree shrew Tupaia belangeri to human hepatitis B virus (HBV) has been demonstrated both in vivo and in vitro. In this study, we show that purified HBV infects primary T. belangeri hepatocyte cultures in a very specific manner, as detected by HBV covalently closed circular DNA, mRNA, HBV e antigen, and HBsAg production. A monoclonal antibody (MAb), MA18/7, directed against the pre-S1 domain of the large HBs protein, which has been shown to neutralize infectivity of HBV for primary human hepatocytes, also blocked infection of primary Tupaia hepatocytes. MAbs against the pre-S2 domain of HBs inhibited infection only partially, whereas an S MAb and polyvalent anti-HBs antibodies neutralized infection completely. Thus, both pre-S1 and S antigens are necessary for infection in the tupaia. Using subviral particles, >70% of primary Tupaia hepatocytes are capable of specific binding of pre-S1-rich HBsAg, showing localization in distinct membrane areas. The data show that the early steps of HBV infection in Tupaia hepatocyte cultures are comparable to those in the human system.     

Alterations in the human immune response to the hepatitis B vaccine among the elderly

The specific binding of hepatitis B (HBs) antigen by lymphocytes from old people immunized with hepatitis B vaccine was explored. For that purpose HBs antigen was combined with fluorescent microspheres, and labeled antigen was allowed to react with lymphocytes from HBs vaccine-responsive or unresponsive people. Lymphocytes from 10 responders and 14 nonresponders were tested for their antigen-binding ability. For controls, lymphocytes were incubated with microspheres bearing human albumin. Lymphocytes from 8 out of 10 responders were able to recognize HBs antigen; for the nonresponders the ratio was 9 out of 14. HBs-binding lymphocytes were B cells but not T lymphocytes. B and T cells from responders and nonresponders were combined and cultivated for 8 days in the presence of HBs antigen, and antibody-producing cells were counted. Neither B cells alone nor B cells plus T cells from nonresponders were able to produce antibody. On the other hand B cells from unresponsive old people produced antibodies when they were cultivated in the presence of HBs antigen and T cells from responsive old people. These data suggest that some elderly individuals who do not produce antibody after in vivo immunization by HBs vaccine do have antibody-producing cells. Instead of a gap in their immune repertoire, these people are suffering from immune dysfunction.     

Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of "a" determinant variants

In chronic hepatitis B (CHB), the persistence of hepatitis B surface antigen (HBs Ag) is sometimes associated with antibodies (Ab) to HBs (anti-HBs). To assess the hypothesis of the selection of HBs Ag immune escape variants in CHB patients, the variability of the HBV S gene was determined for patients persistently carrying both HBs Ag and anti-HBs antibodies and patients solely positive for HBs Ag. We selected 14 patients who presented both markers (group I) in several consecutive samples and 12 patients positive for HBs Ag only (group II). The HBs Ag-encoding gene was amplified and cloned, and at least 15 clones per patient were sequenced and analyzed. The number of residue changes within the S protein was 2.7 times more frequent for group I than for group II patients and occurred mostly in the "a" determinant of the major hydrophilic region (MHR), with 9.52 versus 2.43 changes per 100 residues (P = 0.009), respectively. Ten patients (71%) from group I, but only three (25%) from group II, presented at least two residue changes in the MHR. The most frequent changes in group I patients were located at positions s145, s129, s126, s144, and s123, as described for immune escape variants. In CHB patients, the coexistence of HBs Ag and anti-HBs Ab is associated with an increase of "a" determinant variability, suggesting a selection of HBV immune escape mutants during chronic carriage. The consequences of this selection process with regard to vaccine efficacy, diagnosis, and clinical evolution remain partially unknown.     

A simple purification procedure for hepatitis B surface antigen (HBs) by monoclonal antibody immunosorbent affinity chromatography

Using immobilized monoclonal antibodies (anti-HBs) to hepatitis B surface antigen (HBs) as an immunosorbent affinity column, a simple and effective procedure for HBs purification has been developed. A serum sample containing high titer HBs (i.e. HBe-positive serum) is passed through the column without prior treatment. The HBs is further purified by cesium chloride gradient ultracentrifugation. The recovery of HBs is greater than 70% while the purity is very comparable to those obtained from several isopycnic and rate zonal ultracentrifugation procedures (Dreesman et al., 1972). Over a period of two months, the column was used repeatedly for thirty cycles without any noticeable deterioration.     

IgE antibody against hepatitis B surface antigen in mice

Hepatitis B surface antigen (HBs antigen) was examined for elicitation of IgE production by injection into mice. The Prausnitz-Küstner (PK)-type skin test in the rat was employed for detection of IgE antibody to HBs antigen, because no sufficient purified HBs antigen was available as the challenging antigen for the passive cutaneous anaphylaxis (PCA) test in rats. The positive PK test was considered to be due to IgE antibody, since the active principle was inactivated by heating the sera at 56 C for 30 min, did not bind to protein A and was eliminated by anti-mouse IgE antisera. These data indicate that the PK-type test in rats can be used for detection of mouse IgE antibody when the amount of a test sample is not sufficient for the PCA test in rats.     

Immunogenicity of hepatitis B surface antigen derived from the baculovirus expression vector system: a mouse potency study

A standard mouse potency test was performed to evaluate the immunogenicity of recombinant hepatitis B surface antigen (HBsAg) produced in the baculovirus/insect cell expression system. Groups of NIH Swiss mice were immunized with serial four-fold amounts of either baculovirus-derived HBsAg adsorbed to aluminum sulfate or a commercially available yeast-derived recombinant HBsAg vaccine preparation. Results from these experiments showed that the effective dose of baculovirus- and yeast-derived HBsAg vaccine preparations necessary to seroconvert 50% of the animals were similar. The duration of the antibody response to HBsAg was studied in mice immunized with the highest doses of the two recombinant vaccine preparations 3 and 6 months after injection. No decrease in the anti-HBs response was observed 6 months after injection. No decrease in the anti-HBs response was observed 6 months after immunization with either of the two vaccine preparations. These results indicate that the baculovirus-derived recombinant HBsAg could serve as an alternative vaccine candidate for hepatitis B virus.     

Spontaneous development of anti-hepatitis B virus envelope (anti-idiotypic) antibodies in animals immunized with human liver endonexin II or with the F(ab')2 fragment of anti-human liver endonexin II immunoglobulin G: evidence for a receptor-ligand-like relationship between small hepatitis B surface antigen and endonexin II.

In a previous study, we have identified endonexin II (E-II) on human liver plasma membranes as a specific, Ca(2+)-dependent, small hepatitis B surface antigen (HBsAg)-binding protein. In this article, we describe the spontaneous development of anti-HBs antibodies in rabbits immunized with native or recombinant human liver E-II and in chickens immunized with the F(ab')2 fragment of rabbit anti-human liver E-II immunoglobulin G. Anti-HBs activity was not observed in rabbits immunized with rat liver E-II. Cross-reactivity of anti-E-II antibodies to HBsAg epitopes was excluded, since anti-HBs and anti-E-II activities can be separated by E-II affinity chromatography. The existence of an anti-idiotypic antibody is further demonstrated by competitive binding of human liver E-II and this antibody (Ab2) to small HBsAg, suggesting that Ab2 mimics a specific E-II epitope that interacts with small HBsAg. In addition, it was demonstrated that anti-HBs antibodies developed in rabbits after immunization with intact human liver E-II or in chickens after immunization with F(ab')2 fragments of rabbit anti-human liver E-II immunoglobulin G recognize the same epitopes on small HBsAg. These findings strongly indicate that human liver E-II is a very specific small HBsAg-binding protein and support the assumption that human liver E-II is the hepatitis B virus receptor protein.     

Family diffusion of HBs antigen]

Systematic investigation for HBs antigen and for HBs antibody in donors carrying HBs antigen evidenced a familial contamination more important in children and first cousins than in husband and wife which confirm various previous studies. Moreover, daughters and sisters of antigen carriers show an abnormaly high antigen frequency and especially an abnormally low antibody frequency.     

Prevalence of hepatitis B markers, antibodies to adult T cell leukemia/lymphoma virus and antibodies to human immune deficiency virus in prostitutes in Fukuoka, Japan

Prevalence of hepatitis B (HB) markers, antibodies to human T cell leukemia virus (HTLV-1) and antibodies to human immune deficiency virus 1 (HIV-1) in prostitutes working in Fukuoka city were studied. Sera were collected from 237 prostitutes during January-September, 1986. Among them, 9 (3.8%) were HB virus surface-antigen (HBs Ag) positive, of whom, 3 were HBe antigen positive and the remaining 6 were anti-HBe positive. The positive rate of anti-HBs was 34.2%. The incidence of anti-HTLV-1 in the prostitutes was 5.9%. These incidences are considered to be within the usual range in Kyushu district. No seropositive case for anti-HIV was found.     

Enhanced chemiluminescence ELISA for the detection of antibody to hepatitis B virus surface antigen

A chemiluminescent enzyme linked immunosorbent assay (ELISA) for the detection of antibody to hepatitis B virus surface antigen (anti-HBs) in human serum has been developed. Polystyrene microtitre plates were coated with recombinant, yeast-derived hepatitis B surface antigen (rec-HBsAg). Patient serum samples and appropriate controls were added to the rec-HBsAg-coated wells and incubated to bind anti-HBs. The wells were then washed and a fluorescein isothiocyanate (FITC) conjugate of a human plasma-derived hepatitis B surface antigen (HBsAg) was added. Following incubation and further washing the bound FITC-labelled HBsAg was detected after addition of a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody and assaying for the enzyme. The activity of the HRP was measured using luminol and hydrogen peroxide as substrates and iodophenol as a chemiluminescence enhancer. The luminescence was recorded using a camera luminometer. Preliminary tests have shown the assay to be suitable for the detection of antibody in sera from both vaccinees and also from individuals with a past hepatitis B virus infection. The use of the FITC-anti-FITC system together with the measurement of a chemiluminescence signal makes possible the completion of this assay in a few hours. The assay has been shown to be both specific and sensitive and provides a permanent photographic record.     

A New ELISA to Overcome the Pitfalls in Quantification of Recombinant Human Monoclonal Anti-HBs,GC1102, by Commercial Immunoassays

Several methods for the quantification of human anti-HBs, an antibody to hepatitis B surface antigen (HBsAg), have been developed based on enzyme reaction, chemiluminescence, fluorescence, and radioactivity for application to human serum or plasma. Commercial anti-HBs immunoassay kits use a sandwich method in which a bridge is formed by the anti-HBs between a HBsAg immobilized solid matrix and the labeled HBsAg. However, this direct sandwich enzyme-linked immunosorbent assay (ELISA) is insufficient to accurately evaluate the activity of the human monoclonal anti-HBs, GC1102. As an alternative, we developed an indirect anti-HBs ELISA (anti-HBs qELISA_v.1) that improved detection of anti-HBs. In this current study, we further optimized this indirect method to minimize nonspecific binding of human serum, by employing incubation buffers containing animal serum, Tween 20, skim milk, and a low pH washing buffer. This new and improved method, termed anti-HBs qELISA_v.2, showed accurate quantification of plasma-derived hepatitis B immune globulin (HBIG) and was comparable to results obtained with commercial ELISA (r?=?0.93) and RIA (r?=?0.85) kits. Further, the GC1102 in human serum could be precisely measured using the anti-HBs qELISA_v.2 without limitations of nonspecific binding.     

Comparison of hepatitis B,core, HBc,and hepatitis B antibody,anti HBs,in a presumed low risk donor population

Donors screened by medical social history interview negative for high risk behavior or communicable disease history, but subsequently exhibiting reactive serological markers, emphasize importance of duel safe guarding factors for determining donor suitability. This report examines a relationship between two immunoabsorption assay tests, hepatitis B core (HBc) antibody, a required food and drug administration (FDA) test, and hepatitis B antibody (anti HBs), non-required test. Reactive serology results, 129 cases, 3,581 donors (2008–2012) for HBc as the only initially positive serological marker were subjected to anti HBs testing in this history pre-screened donor population. Enzyme linked immunoabsorption assay kits hepatitis B, core and antibody, were used in this study. All samples were initially tested for human immunodeficiency virus, hepatitis B, and hepatitis C, utilizing nucleic acid testing and antigen antibody immunoabsorption assay. Testing was performed by a FDA-registered CLEA-certified reference laboratory. Samples were deceased donor blood samples and a limited number of pre-mortem samples, separated, stored and analyzed according to manufacturer recommendation and FDA regulations. 129 reactive HBc only samples, were subsequently tested for anti HBs. Of these 129, 94 were found to be reactive for anti HBs. This represented 72 % of samples tested for antibody, a higher percentage than anticipated for a medical history negative, low risk population.     

Coincident hepatitis B surface antigen and antibodies of different subtypes in human serum.

Three patients with simultaneously detectable hepatitis B surface antigen (HBsAg) and antibody (anti-HBs) in their sera were studied for subtypes of HBsAg and anti-HBs. In each case anti-HBs was found to be directed to a different subtype than that of the circulating HBsAg, indicating that reinfection (or simultaneous infection) with a second subtype occurred.     

l-Proline reduces IgG dimer content and enhances the stability of intravenous immunoglobulin (IVIG) solutions

Liquid intravenous immunoglobulin (IVIG) products offer improved convenience in preparation but often lack sufficient stability to allow room temperature storage. Furthermore, clinical tolerability may be affected due to formation of idiotype/anti-idiotype IgG dimers and/or aggregates. Here we report on the development of a 10% IVIG formulation with optimized stability achieved by the use of l-proline. The stability of concentrated liquid IVIG was strongly pH dependent. Aggregate formation, yellowish discoloration of the solution and loss of anti-hepatitis B surface antigen (HBs) antibody activity was minimal at intermediate pH (pH 4.8–5.3). Fragmentation of IgG was highest at low pH (pH 4.1). Idiotype/anti-idiotype IgG dimer formation was highest at neutral pH and was reduced with decreasing pH. The presence of l-proline further improved stability by inhibiting protein aggregation, reducing loss of anti-HBs antibody activity and decreasing coloring, particularly compared with glycine formulations. The IgG dimer content was up to 30% lower in solutions containing l-proline compared with those containing glycine or other stabilizers. In conclusion, a weakly acidic pH of approximately 5 and l-proline as stabilizer are optimal conditions for long-term stability of a liquid IVIG. l-proline, an amphiphilic, naturally occurring amino acid, is superior to glycine in restricting IgG dimer formation.     

Hepatocellular carcinoma in Japan and its linkage to infection with hepatitis C virus

Hepatitis B and C viruses are closely related to the development of liver cirrhosis and hepatocellular carcinoma. Recently these two viral agents were found to be the major causative agents of hepatocellular carcinoma in Japan. An increase in the number of HCV antibody-positive patients, but a decrease in the number of HBs antigen-positive patients with hepato cellular carcinoma has been noted over the last 15 years. In the late 1980s about 70% of the patients with hepatocellular carcinoma were found to be positive for HCV antibody and 24% for HBs antigen. Presence of several subtypes of HCV was reported. Hypervariable region (HVR) in a putative envelope, gp70, was detected within the same subtype. Variability of HVR seemed to be the result of spontaneous mutation caused after infection. Such changes with time in the sequence of the HCV genome in the blood of patients with type C hepatitis are likely to be due to immunological surveillance by the host.     

Hepatitis B surface antigen (HBsAg) infection in a hemodialysis unit. II. Factors affecting host immune response to HBsAg.

Serum from 86 hemodialysis patients, 105 healthy hospital staff "at risk" and 160 regular hospital staff was screened for hepatitis B surface antigen (HBsAg) and antibody (anti-HBs). The combined prevalence of HBsAg and anti-HBs was higher in the staff of the artificial kidney unit (57.7%) than in the hemodialysis patients (33.7%). The healthy subjects with HBsAg infection responded significantly more often by producing anti-HBs compared with the hemodialysis patients. Twelve of 29 (41.4%) hemodialysis patients with HBsAg infection produced anti-HBs, while 17 (58.6%) remained positive for HBsAg. This differential response could not be attributed to age, sex, time spent undergoing hemodialysis, delayed cutaneous reactivity or response to phytohemagglutinin (PHA) or pokeweed mitogen (PWM). However, a much larger proportion of patients with HBsAg than with anti-HBs had previously received blood transfusions (88.2% v. 33.3%). Our results indicate that development of the chronic HBsAg carrier state or production of anti-HBs in uremic patients may be influenced by the route of immunization or the dose of antigen, or both. Although uremic patients maintain normal in vitro response to PHA and PWM, they may have depressed immunity in vivo because of a decreased total number of T-lymphocytes.     

The immunogenicity of recombinant LC16mO vaccinia virus harboring HBsAg gene in mice

It was found that hepatitis B surface antigen (HBsAg) expressed by recombinant vaccinia virus (RVV), rProHBmO143, harboring HBsAg gene was immunologically similar to plasma-derived HBsAg and immunogenicity of the rProHBmO143 was possible to evaluate by the skin scarification (SS) method using BALB/c mice. When we compared the immunogenicity of 10(8) TCID50 of the rProHBmO143 by the SS method with that of 0.125 ml of the plasma-derived hepatitis B vaccine (HB vaccine) given by intraperitoneal inoculation, the anti-HBs antibody eliciting ability of its RVV was almost the same as that of the HB vaccine with maintenance of high antibody titers, and the antibody responses rose further by re-inoculation in association with HB vaccine, especially by using its RVV as a priming. Also, no virus was recovered from the liver, spleen or brain of the mice inoculated with rProHBmO143 by the SS method. Furthermore, in mice inoculated with rProHBmO143 and then inoculated with RVV harboring Japanese encephalitis virus (JEV) gene 24-weeks later, no effect was recognized on duration of anti-HBs antibody persistence while anti-JEV antibody is being produced. These results suggest that the rProHBmO143 is likely to become a practical live vaccine; a different immunization schedule to protect against hepatitis B virus and two or more kinds of RVV vaccines may be usable for the same animal or humans at intervals of some years.     

乙肝病毒表面抗原和抗体双阳性者中病毒S区基因序列分析

为了解乙肝病毒 (HBV)表面抗原和抗体双阳性患者中病毒的基因型及其HVBS区是否有变异。用放射免疫试剂检测HBsAg阳性样品中的抗 HBs抗体 ,用聚合酶链反应法检测双阳性样品中的HBVDNA ,然后对阳性样品进行克隆和基因序列分析 ,并将所得序列与HBV不同基因型的代表株进行比较分析。结果显示 389例HBsAg阳性样品中有 10例为抗HBs抗体阳性 ;该 10例双阳性样品中有 5例为HBVDNA阳性 ;序列分析显示该 5株HBV均为B基因型 ,其中 4株为adw亚型 ,1株为adr亚型 ;其中有 2株在S区的“a”决定簇的氨基酸发生了变异