Visualization and identification of hepatitis C viral particles by atomic force microscopy combined with MS/MS analysis

A possibility of detection and identification of hepatitis C viral (HCV) particles by atomic force microscopy (AFM) in combination with mass spectrometry (MS) has been investigated. The AFM/MS approach is based on two technologies: 1. AFM-biospecific fishing that allows to detect, concentrate from solution and to count protein complexes on the surface of a AFM-nanochip; 2. mass spectrometric identification of these complexes. AFM-biospecific fishing of HCVcoreAg from solution was carried onto the surface of AFM-nanochips with immobilized anti-HCVcoreAg. It was shown that HCVcoreAg/anti-HCVcore_im complexes were formed on AFM-nanochips in quantity sufficient for their subsequent mass spectrometric identification. Thus, the AFM/MS approach allows to identify fragments of hepatitis C virus fished on the surface of AFM-nanochip from serum.     

Detection of hepatitis B virus surface antigen with the use of an optical biosensor

The detection of hepatitis B virus surface antigen (HBsAg) with the use of a model IAsys+ two-channel optical biosensor is based on the registration of interaction between anti-HBs monoclonal antibodies forming the surface layer of the biochip of the biosensor cuvette and blood serum HBsAg. For the first time a two-channel optical biosensor has been used for the detection of HBsAg in blood serum samples. The comparative analysis of the detection of HBsAg by two methods, viz. with the use of an optical biosensor and the enzyme immunoassay, has demonstrated lower sensitivity, but higher specificity of the detection of this antigen by means of a model IAsys+ biosensor with the biochip, prepared in the process of the work. The main advantages of the biosensor detection lie in the registration of interaction in real time without introducing special markers into the molecules under study.     

Effect of conditions of monoclonal antibody adsorption on antigen-binding activity

The dependence of the antigen-binding activity of immobilized antibodies on pH of a saturating buffer has been investigated. We analyzed 28 monoclonal antibodies (MCAs) produced by various hybridomas to three virus antigens, i.e., the nuclear p23 protein of hepatitis C virus (C core protein p23), p24 protein of HIV 1, and the surface antigen of hepatitis B virus (HBsAg). Antibodies were adsorbed on the surfaces of immune plates in acidic (pH 2.8), neutral (pH 7.5), and alkaline (pH 9.5) buffers. The binding of labeled antigens, i.e., biotinylated or conjugated with horseradish peroxidase, with immobilized antigens was tested. It was shown that 10 out of 28 analyzed MCAs (36%) considerably better preserved their antigen-binding activity if their passive adsorption was carried out on the surface of polystyrene plates in an acidic buffer (pH 2.8). This approach allowed constructing a highly sensitive sandwich method for HBsAg assay with a minimal reliably determined antigen concentration of 0.013–0.017 ng/ml. The described approach may be recommended for the optimization of sandwich methods and solid-phase competitive methods.     

A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum

Current clinically assays, such as enzyme‐linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high‐throughput analysis. A novel assay based on magnetic beads and time‐resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti‐HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium‐labeled anti‐HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02–700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra‐ and interassay coefficients of variation were 4.7–8.7% and 3.8–7.5%, respectively. The performance of this assay was further assessed against a well‐established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X – 0.017, R = 0.989). In the current study, we demonstrated that this novel time‐resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high‐throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus. Copyright © 2013 John Wiley & Sons, Ltd.     

Evidence for hepatitis C viral infection in patients with primary hepatocellular carcinoma.

In testing for antibodies to the hepatitis C virus (anti-HCV) in 112 patients with primary hepatocellular carcinoma, 10 of 33 white patients (30%) and 15 of 79 Asian patients (19%) had a positive response to the antibody. The antibody profile to individual hepatitis C viral antigens and the presence of circulating hepatitis C viral RNA were determined in the 25 patients. The anti-HCV antibodies most frequently detected were toward the antigens from the core (C22) and NS3 regions. Serum hepatitis C viral RNA was present in 17 of the 25 patients (68%), and these patients tended to have serum levels of alanine and aspartate aminotransferases higher than those patients without viremia (136 +/- 22 U per liter versus 64 +/- 11 U per liter and 161 +/- 26 U per liter versus 79 +/- 14 U per liter, respectively, both P < .05). Of the 15 Asian patients with hepatocellular carcinoma and anti-HCV, 4 (27%) had coexisting hepatitis B surface antigen (HBsAg) and 13 (87%) had antibodies to either hepatitis B core or surface antigen. Of the 10 white patients with anti-HCV, however, only 1 (10%) had hepatitis B virus antibodies (P < .01). Among 4 Asian patients with coexisting anti-HCV and HBsAg, 1 was found to have serum hepatitis B viral DNA and the other 3 had hepatitis C viral RNA. A history of blood transfusion was obtained from 12 of the 25 patients with anti-HCV (48%); 20 (80%) had coexisting cirrhosis. Our findings support the hypothesis that hepatitis C virus is an important etiologic agent in the development of primary hepatocellular carcinoma in both white and Asian patients in the United States.     

CpG Oligodeoxynucleotides with Hepatitis B Surface Antigen (HBsAg) for Vaccination in HBsAg-Transgenic Mice

DNA motifs containing unmethylated CpG dinucleotides within the context of certain flanking sequences enhance both innate and antigen-specific immune responses, due in part to the enhanced production of Th1-type cytokines. Here we explored the ability of CpG-containing oligodeoxynucleotides combined with recombinant hepatitis B surface antigen (HBsAg) to induce Th1 responses in mice that are transgenic for this antigen and that represent a model for asymptomatic hepatitis B virus chronic carriers. This was compared to hepatitis B virus-specific DNA-mediated immunization, which we have previously shown to induce the clearance of the transgene expression product and the down-regulation of hepatitis B virus mRNA in this transgenic mouse lineage. In control nontransgenic C57BL/6 mice, three immunizations with HBsAg and CpG triggered the production of anti-HBs antibodies and of HBs-specific T cells that secrete gamma interferon but do not display any HBsAg-specific cytotoxic activity. In the HBsAg-transgenic mice, immunization with HBsAg and CpG oligodeoxynucleotides, but not with CpG alone, induced the clearance of HBsAg circulating in the sera, with a concomitant appearance of specific antibodies, and was able to regulate the hepatitis B virus mRNA constitutively expressed in the liver. Finally, adoptive transfer experiments with CD8(+) T cells primed in C57BL/6 mice with HBsAg and CpG oligodeoxynucleotide-based immunization show that these cells were able to partially control transgene expression in the liver and to clear the HBsAg from the sera of recipient transgenic mice without an antibody requirement. CpG oligodeoxynucleotides motifs combined with HBsAg could therefore represent a potential therapeutic approach with which to treat chronically infected patients.     

Immunoenzyme analysis for determining class G and M antibodies to HBsAg

A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results.     

Organ-specific expression of hepatitis B surface antigen in potato

Summary The gene encoding the hepatitis B surface antigen (HBsAg) under the control of cauliflower mosaic virus 35S-promoter was constructed and expressed in transgenic potato plants. The HBsAg expression were measured by ELISA kit containing monoclonal antibodies. The amount of HBsAg in roots was found 5–10 fold higher than in leaf tissues     

Construction and characterization of hybrid hepatitis B antigen particles carrying a poliovirus immunogen

The hepatitis B surface antigen (HBsAg) has the unique property of assembling with cellular lipids into spherical or elongated particles of 22 nm diameter which are secreted by mammalian cells expressing HBsAg. We have studied the structural requirements for particle formation and secretion by creating in-phase insertions into different regions of the S gene of the hepatitis B virus, coding for HBsAg. Modified genes were integrated into an appropriate vector and expressed in mouse L cells. Various single and double inserts in the two major hydrophilic domains of HBsAg were compatible with particle synthesis and secretion. The level of secretion was influenced by the length of the insert, its primary structure, and the site of insertion into the HBsAg molecule. One of the inserted sequences was a synthetic DNA fragment encoding a continuous type 1 poliovirus neutralization epitope (the C3 epitope). Mammalian cells expressing the modified hepatitis B virus S gene secreted hybrid particles carrying the poliovirus antigen. The hybrid polio-HBsAg particles reacted with a monoclonal antibody specific for the C3 epitope and induced poliovirus neutralizing antibodies at low, but significant, titers in mice and at high titers in rabbits. However, the immune response to HBsAg was weaker to hybrid particles than to unmodified HBsAg particles. By cotransfection with two different plasmids carrying either modified or unmodified genes, we obtained phenotypically mixed particles containing both polio-HBsAg and HBsAg molecules. Inoculated into rabbits, the mixed particles induced high antibody titers against both poliovirus and HBsAg.     

Hepatitis B virus DNA and e antigen in serum from blood donors in the United Kingdom positive for hepatitis B surface antigen.

Serum samples from 214 blood donors in the United Kingdom who were carriers of hepatitis B surface antigen (HBsAg) were examined for hepatitis B virus deoxyribonucleic acid (DNA) by DNA:DNA hybridisation and for hepatitis B e antigen (HBeAg) and its antibody. One fifth of the donors carried infectious virus in their circulation. The presence of hepatitis B virus DNA correlated well with that of HBeAg, although hepatitis B virus DNA was found in five serum samples that were negative for HBeAg. It is concluded that analysis of serum samples for hepatitis B virus DNA by hybridisation should be the method of choice for determining whether carriers of HBsAg are infectious.     

Vaccine- and hepatitis B immune globulin-induced escape mutations of hepatitis B virus surface antigen

Hepatitis B virus surface antigen (HBsAg) vaccination has been shown to be effective in preventing hepatitis B virus (HBV) infection. The protection is based on the induction of anti-HBs antibodies against a major cluster of antigenic epitopes of HBsAg, defined as the 'a' determinant region of small HBsAg. Prophylaxis of recurrent HBV infection in patients who have undergone liver transplantation for hepatitis B-related end-stage liver disease is achieved by the administration of hepatitis B immune globulins (HBIg) derived from HBsAg-vaccinated subjects. The anti-HBs-mediated immune pressure on HBV, however, seems to go along with the emergence and/or selection of immune escape HBV mutants that enable viral persistence in spite of adequate antibody titers. These HBsAg escape mutants harbor single or double point mutations that may significantly alter the immunological characteristics of HBsAg. Most escape mutations that influence HBsAg recognition by anti-HBs antibodies are located in the second 'a' determinant loop. Notably, HBsAg with an arginine replacement for glycine at amino acid 145 is considered the quintessential immune escape mutant because it has been isolated consistently in clinical samples of HBIg-treated individuals and vaccinated infants of chronically infected mothers. Direct binding studies with monoclonal antibodies demonstrated a more dramatic impact of this mutation on anti-HBs antibody recognition, compared with other point mutations in this antigenic domain. The clinical and epidemiological significance of these emerging HBsAg mutants will be a matter of research for years to come, especially as data available so far document that these mutants are viable and infectious strains. Strategies for vaccination programs and posttransplantation prophylaxis of recurrent hepatitis need to be developed that may prevent immune escape mutant HBV from spreading and to prevent these strains from becoming dominant during the next decennia.     

Hepatitis B infection among patients attending a sexually transmitted diseases clinic in Rio de Janeiro, Brazil

Hepatitis B virus (HBV) has a low endemicity in Rio de Janeiro, Brazil. Sexual transmission must play an important role in this virus, but the prevalence and risk factors have never been properly investigated. The aim of this paper is to determine the prevalence and risk factors for HBV infection in patients attending a Sexually Transmitted Diseases Clinic of the Universidade Federal Fluminense, from the State of Rio de Janeiro, Brazil. In a retrospective study, HBV seroprevalence was investigated in 440 patients. Serum of each patient was assayed for antibodies against hepatitis B core antigen (anti-HBc), hepatitis B surface antigen (HBsAg) and antibodies against hepatitis B surface antigen (anti-HBs). Demographic and risk factor data were extracted from clinic notes. The overall seroprevalence of exposure markers for HBV (anti-HBc, HBsAg and anti-HBs) were 13%, 3.4% and 8.5% respectively. Homo/bisexual behaviour, anal intercourse, HIV infection, positive serology for syphilis and blood transfusion were predictors of the HBV exposure. Among demographic data, age and place of birth were associated with the anti-HBc seropositivity.     

Determination of antibody to hepatitis B core antigen by means of immune adherence hemagglutination.

A simple and rapid method utilizing immune adherence hemagglutination has been developed for the detection of antibodies to hepatitis B core antigen (anti-HBc). Hepatitis B core antigen (HBcAG) was prepared from Dane particles that had been isolated from plasma of asymptomatic antigen carriers. The method was specific and about 10 times more sensitive than the conventional complement-fixation method. A total of 215 serum samples obtained from healthy blood donors were surveyed for HBsAG and anti-HEc, as well as for hepatitis B surface antigen (HBsAg) and antibody to HBsAG (anti-HBs). Anti-HBc was found in 36 serum samples, at a prevalence rate higher than that of anti-HBs (31/215)...     

IgM antibody against hepatitis B core antigen (IgM anti-HBc): diagnostic and prognostic significance in acute HBsAg positive hepatitis.

IgM antibody against hepatitis B core antigen (IgM anti-HBc), a marker of recent hepatitis B virus infection, was sought by radioimmunoassay in sera diluted 1/4000 from 376 patients presenting to four centres in Italy with acute, apparently type B hepatitis (hepatitis B surface antigen (HBsAg) positive). In 320 patients (85%) a positive IgM anti-HBc test result confirmed that hepatitis was due to primary infection with hepatitis B virus. In the remaining 56 patients absence of the IgM marker indicated that they were previously unrecognised long term carriers of HBsAg. Further serum analysis often showed delta infection and occasionally hepatitis A or cytomegalovirus infection as the true cause of their illness. After six to eight months circulating HBsAg persisted in 38 of 45 patients (84%) without IgM anti-HBc but in only six of 150 patients (4%) with the IgM antibody (p less than 0.0001). A negative IgM anti-HBc test result in patients with acute HBsAg positive hepatitis points to a factor other than hepatitis B virus as the cause of the liver damage and predicts the carriage of HBsAg.     

Soluble HLA class I and HLA class II antigens in the blood of HIV infected patients

The levels of HLA, class I, antigen and HLA-DR antigen in the blood sera of HIV-infected persons were determined by the enzyme immunoassay. The levels of soluble antigens HLR-DR and of HLA, class I, in serum samples containing only antibodies to HIV were elevated, respectively, 1.8- and 1.3-fold in comparison with the norm. The level of soluble HLA-DR antigen in samples containing the markers of other infections (HBsAg, antibodies to hepatitis C virus, anti-IgM antibodies to cytomegalovirus) was significantly higher in comparison with samples containing only antibodies to HIV and serum samples from healthy donors (p < 0.05). The concentration of soluble HLA antigen, class I, in the samples containing antibodies to HIV and markers of other infections was also elevated, but the statistically authentic increase in comparison with the normal level was observed only in the presence of the markers of HIV infection, hepatitis C and cytomegalovirus infection.     

Prevalence and determinants of antibodies to hepatitis C virus and markers for hepatitis B virus infection in patients with HIV infection in Aquitaine. Groupe d'Epidémiologie Clinique du SIDA en Aquitaine.

OBJECTIVE: To evaluate the prevalence of antibodies to hepatitis C virus and serological markers for hepatitis B virus infection in patients with HIV. DESIGN: Cross sectional survey. SETTING: Aquitaine, southwestern France, 1991-94. SUBJECTS: 1935 HIV positive patients seen at least once since June 1991. MAIN OUTCOME MEASURES: Presence of antibodies to hepatitis C virus were detected by second or third generation enzyme linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA) and markers for hepatitis B virus detected by ELISA. RESULTS: The prevalence was 42.5% (823) for antibodies to hepatitis C virus, 56.4 (507) for antibodies to hepatitis B core antigen, 6.9% (133) for hepatitis B surface antigen, 30.2% (584) for antibodies to hepatitis B core and surface antigen with no detectable surface antigen, 26.2% (507) for antibodies to core antigen only, and 4.8% (92) for antibodies to surface antigen only. The prevalence of antibodies to hepatitis C virus was 86.1% (726/843) in subjects who had bloodborne HIV infection and 7.3% (66/899) in those with sexually acquired infection. The prevalence of markers for hepatitis B was higher among homosexuals than in the other groups of patients, except for antibodies to surface antigen alone. The relation between markers for hepatitis B and hepatitis C virus was negative among men but positive among women. CONCLUSIONS: The results favour the hypothesis that hepatitis C virus is sexually transmitted much less commonly than either HIV or hepatitis B virus.     

Synthesis in yeast of hepatitis B virus surface antigen modified P31 particles by gene modification

The pre-S2 portion of hepatitis B virus surface antigen P31 gene was modified to make gene products resistant to trypsin-like proteases in Saccharomyces cerevisiae. The coding sequence for 6 amino acids (Ser44 - Thr49) including Arg48 was removed, and the altered gene was inserted into an expression vector. The modified HBsAg P31 (M-P31c) gene products, consisting of GP37 and GP34, formed particles having both HBsAg antigenicity and polymerized-albumin receptor activity. Since the M-P31c particles can elicite two kinds of protective antibodies against hepatitis B virus, anti-S and anti-pre-S2 antibodies, the M-P31c particles are expected to be potentially effective to S-nonresponders.     

Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope.

Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d.     

Immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis B and human immunodeficiency viruses

A synthetic chimeric gene, TBI-HBS, encoding the immunogenic ENV and GAG epitopes of human immunodeficiency virus (HIV-1) and the surface protein antigen (HBsAg) of hepatitis B virus (HBV), was expressed in tomato plants. Tomato fruits containing the TBI-HBS antigen were fed to experimental mice and, on days 14 and 28 post-feeding, high levels of HIV- and HBV-specific antibodies were present in the serum and feces of the test animals. Intraperitoneal injection of a DNA vaccine directing synthesis of the same TBI-HBsAg antigen boosted the antibody response to HIV in the blood serum; however, it had no effect on the high level of antibodies produced to HBV.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.