Hepatitis B virus e antigen specific epitopes and limitations of commercial anti-HBe immunoassays

Current commercial hepatitis B virus (HBV) anti-HBe immunoassays are designed so that anti-HBe is detectable only in the absence of excess HBeAg. Recently, with the use of direct anti-HBe assays, anti-HBe was detected in individuals who had been seropositive for several years for HBeAg [Maruyama et al. (1993) J. Clin. Invest. 91:2586-2595]. Although anti-HBe seroconversion does not necessarily indicate subsequent HBeAg clearance, the ability to detect earlier anti-HBe seroconversion could have clinical significance for monitoring patients undergoing HBV immunotherapy (e.g., alpha interferon therapy). Because the HBeAg and the HBcAg share 149 amino acids, an anti-HBe assay must distinguish anti-HBe from anti-HBc antibodies. Although the HBV HBeAg and HBcAg display distinct immunogenic determinants, much remains unknown regarding the complete epitope spectrum specific to each antigen. The goal of this study was 3-fold. The first objective was to identify HBeAg specific linear epitopes. The second objective was to design an anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg. The third objective was to characterize early anti-HBe seroconversion antibodies. The major linear epitope residing in the HBeAg amino acid sequence was mapped and 2 novel minor epitopes (delta, gamma) which appear to be HBeAg specific have been identified. An anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg was designed. Finally, it was found that early anti-HBe seroconversion antibodies appear to be conformational, whereas later seroconversion, more typically associated with the clearance of HBeAg, is characterized by the presence of antibodies to the linear HBeAg epitopes.     

Inexpensive Designer Antigen for Anti-HIV Antibody Detection with High Sensitivity and Specificity

A novel recombinant multiepitope protein (MEP) has been designed that consists of four linear, immunodominant, and phylogenetically conserved epitopes, taken from human immunodeficiency virus (HIV)-encoded antigens that are used in many third-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture, and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium(III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion, and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV enzyme immunoassay (EIA) kits. The careful choice of epitopes, high epitope density, and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium(III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity.Human immunodeficiency virus (HIV) is a lentivirus of the family Retroviridae, whose members characteristically have an RNA genome within a capsid and a lipid envelope. HIV infection induces a profound immune dysfunction, with abnormalities in every arm of the immune system, resulting in AIDS (5). In 2007, there were 2.7 million new HIV infections and 2 million HIV-related deaths. Globally, there were an estimated 33 million people living with HIV in 2007. India is one of the largest and most populated countries in the world, with a population of over 1 billion. Of this number, it is estimated that around 2.4 million Indians were living with HIV in 2007 (26). The genes of HIV are located in the central region of the proviral DNA and encode at least nine proteins. These proteins are divided into three classes: the major structural proteins (Gag, Pol, and Env), the regulatory proteins (Tat and Rev), and the accessory proteins (Vpu, Vpr, Vif, and Nef) (11). The gag gene of HIV type 1 (HIV-1) encodes a polyprotein precursor, p55, which is cleaved by the virus-encoded protease into three proteins, p24, p17, and p15. Linear B-cell epitopes have already been identified within p24 (14). The antigen p24 is of special significance because of its ability to be expressed first in body fluids after HIV-1 infection. The linear immunodominant epitope of p24 serves as an important diagnostic intermediate to detect antibodies to HIV-1 in human sera (23). The envelope glycoproteins (gp), gp41 of HIV-1 and gp36 of the closely related HIV-2, are highly immunogenic and are important diagnostic intermediates for the detection of antibodies to these viruses in human sera (17, 24). HIV-1 comprises three lineages, denoted M, N, and O (22). HIV-2 and divergent forms have been detected in West African or West Africa-related patients with AIDS (7-9). Several enzyme immunoassay (EIA)-based diagnostic kits are available on the market for the detection of antibodies to HIV in human sera. These anti-HIV EIA kits use synthetic peptides and/or recombinant proteins mainly from the envelope gp of HIV-1 group M, HIV-1 group O, and HIV-2. The fourth-generation kits also have antibodies to p24 antigen. The requirement of multiple peptides and/or multiple recombinant proteins for reliable diagnosis of HIV infections adds to the cost of these EIA kits. The high cost of anti-HIV EIA kits becomes prohibitive for routine use in many developing countries, precluding early detection and prevention of new infections (18, 25, 27). We have designed a single recombinant multiepitope protein (MEP) antigen, consisting of several immunodominant, linear, and conserved virus-specific epitopes from structural proteins of HIV-1 and HIV-2. DNAs encoding these epitopes have been assembled in tandem in a single open reading frame, with intervening sequences encoding flexible linkers, and expressed in Escherichia coli. A polyhistidine tag has also been included which allows for facile purification of recombinant MEP by Ni-NTA chromatography. The purified protein has been used as the coating antigen for developing an anti-HIV indirect immunoassay. We have evaluated the performance of this assay with that of other multiple-antigen-based immunoassay kits currently available on the market, using well-characterized commercially available serum panels.     

Evaluation of a Recombinant Multiepitope Peptide for Serodiagnosis of Toxoplasma gondii Infection

Detection of Toxoplasma gondii infection with sensitive and specific methods is a key step in the prevention and treatment of toxoplasmosis. Among the available diagnostic tests, serology is commonly used. Although serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. There is therefore a real need to acquire specific and effective recombinant antigens for the serodiagnosis of T. gondii infection. In this study, a multiepitope peptide was designed and successfully expressed in Escherichia coli, and then IgG and IgM enzyme-linked immunosorbent assays (ELISAs) were developed and evaluated. Our results showed that the new multiepitope antigen is one of the most promising recombinant antigens which could be used in routine screening of human toxoplasmosis.     

Surveillance for hepatitis B surface antigen mutants

Hepatitis B viral (HBV) mutants can emerge in patients as a result of selection pressure from treatment options. Some mutations that occur in the immunodominant "a" determinant of hepatitis B surface antigen (HBsAg) can present as false negative results in HBsAg immunoassays. The mutation position in HBsAg and the type of mutation impacts immunoassay performance. HBsAg mutants will continue to emerge in response to selection pressure, therefore an appropriate HBV immunoassay-testing algorithm needs to be established to ensure their detection. Mutant surveillance programs can also contribute to our understanding of the changing epidemiology of HBV infection.     

New assays for quantitative determination of viral markers in management of chronic hepatitis B virus infection.

We performed a quantitative study of serum hepatitis B virus (HBV) markers, including new parameters such as pre-S1 antigen (Ag), pre-S2 Ag, and anti-HBx, in 88 chronic hepatitis B surface antigen (HBsAg) carriers. New IMx assays for HBsAg and immunoglobulin M (IgM) anti-HBc detection were also used. The population studied was composed of 65 chronic hepatitis cases (40 positive for hepatitis B antigen [HBeAg] and 25 positive for anti-HBe) and 23 anti-HBe-positive, asymptomatic HBsAg carriers. Serum HBsAg levels detected by IMx were higher in HBeAg-positive than in anti-HBe-positive HBsAg carriers (all patient subgroups included) and correlated with the serum HBV DNA level (P = 0.0001). Both pre-S1 and pre-S2 Ags were detected by enzyme immunoassays in almost all HBsAg carriers. Both pre-S1 and pre-S2 Ag titers correlated positively with the serum HBsAg concentration (P = 0.0001), but only the pre-S1 Ag titer correlated with the level of serum HBV DNA (P = 0.02). The detection of low levels of IgM anti-hepatitis B core (anti-HBc) antibodies by IMx was associated with the presence of liver disease (P = 0.05) but not with the level of viral replication. The prevalence of anti-HBx antibodies detected by the enzyme immunoassay was slightly, although not significantly, higher in patients with high levels of HBV DNA (greater than 100 pg/ml) than in patients without detectable HBV DNA (P = 0.16). In anti-HBe-positive chronic HBsAg carriers, the quantitative detection of serum HBV DNA, pre-S Ag titers, and IgM anti HBc allowed us to predict which patients suffered from chronic liver disease and/or supported viral replication (P < 0.05). In a follow-up study of eight patients undergoing antiviral therapy, the clearance of both pre-S1 Ag and HBV DNA was associated with a subsequent clearance of HBV. Therefore, the quantitative determination of HBV DNA, pre-S Ags, IgM anti-HBc may prove useful for the decision to use and the monitoring of antiviral therapy, especially in anti-HBe-positive HBsAg carriers.     

Evaluation of a new rapid test for the combined detection of hepatitis B virus surface antigen and hepatitis B virus e antigen

There are about 350 million chronic hepatitis B virus (HBV) carriers worldwide. A proactive approach to the management of this disease is likely to reduce the morbidity and mortality caused by HBV. This study aimed to evaluate the diagnostic performance of a novel tool for discriminating between infected and noninfected subjects, the hepatitis B sAg/eAg test (Binax Inc., Portland, Maine). The test is designed to rapidly and accurately detect both the HBV surface antigen (HBsAg) and the HBV e antigen (HBeAg). A cohort of 942 subjects was tested. The serum clinical sensitivity of the hepatitis B sAg/eAg test was 99.75 and 96.37% for HBsAg and HBeAg, respectively. Serum clinical specificity was 99.32% for HBsAg and 98.99% for HBeAg. Analytical sensitivity was satisfactory for the purposes of population screening. Visual evaluation showed that the test signals were stable for at least 3 h after the recommended evaluation time. No interference or cross-reactivity was observed with known interfering substances and virologic markers. These results indicate that the hepatitis B sAg/eAg test is well suited to the accurate detection of HBV carriers. In addition to the good clinical specificity and sensitivity of this test, its stability and user-friendly design mean that a correct performance, even under field conditions, is highly likely. Consequently, the hepatitis B sAg/eAg test has the potential to identify subjects who require HBV vaccination (HBsAg(-) and HBeAg(-)) and HBV-infected individuals who might benefit most from antiviral therapy (HBsAg(+) and HBeAg(+)).     

乙肝病毒前S2抗原发光酶免疫方法的临床应用

建立乙肝病毒前Ｓ２抗原发光酶免疫方法，检测ＨＢＶ感染者血清，肝组织及其细胞器中的前Ｓ２，结果与ＨＢｅＡｇ，ＨＢｃＡｇ，ＨＢＶ ＤＮＡ等复制指标一致，某些肝内ＨＢＶ ＤＮＡ阴性者ＰＳ２阳性。这提示前Ｓ２是ＨＢＶ活跃复制的指标。前Ｓ２在肝内主要存于微粒体和核糖体中。前Ｓ２抗体阳性，与肝细胞破坏有关，急性和慢迁肝预示恢复，慢活肝，肝硬化和肝癌预示恶化。本法集发光反应的快速敏感及免疫反应的特异性于一身，敏     

前列腺特异抗原化学发光免疫分析方法的建立及初步应用

建立的前列腺特异抗原(PSA)化学发光免疫分析(CLIA)使用2株抗PSAMcAb,一株McAb包被微孔板,另一株McAb与HRP连接。底物是鲁米诺/H2O2/对碘苯酚系统的双抗体夹心一步法,测定范围0.5～64ng/mL,最低检出值为0.13ng/mL,平均回收率为100.6%,批内和批间CV分别小于8.3%和11.5%,正常参考值小于3.5ng/mL。1000份血清临床考核,与参比试剂盒临床符合率一致,具有同等的临床诊断价值。     

Correlation of HBV DNA and monoclonal reactivity to HBsAg in serum of patients with HBV infection

Hepatitis B virus (HBV) DNA hybridization assay, a monoclonal radioimmunoassay (M-RIA) for hepatitis B surface antigen (HBsAg) and conventional polyclonal immunoassays for HBV associated antigens were used to study sera from patients on dialysis and with acute hepatitis B. HBV DNA was detectable in hepatitis B e antigen (HBeAg) negative patients with acute hepatitis but not in HBsAg+ HBeAg- dialysis patients. In acute hepatitis, HBsAg immunoreactivity by M-RIA could still be detected even though a commercial immunoassay for HBsAg, the AUSRIA II, and the HBV DNA assay were no longer positive. Unlike in acute HBV infection, serum HBV DNA was detectable in dialysis patients who were AUSTRIA II negative but M-RIA positive. Serial determination of HBsAg by M-RIA and HBV DNA revealed episodes of HBV DNA positivity months after both the HBsAg was no longer positive by polyclonal immunoassay. Thus, the M-RIA for HBsAg and the molecular hybridization technique for HBV DNA are sensitive and specific assays for the identification of potentially infectious individuals who would not have been characterized as such based on the results of conventional polyclonal immunoassays.     

Detection of an antigen (MY4) common to M. tuberculosis and M. leprae by 'tandem' immunoassay

A novel 'tandem' immunoassay for the detection of mycobacterial antigen was devised using a monoclonal antibody (ML 34) both as solid phase 'capture' and as the 125I- or enzyme-labelled 'tracer' antibody. This antibody binds to the repeating epitopes (MY4b) of a water-soluble protease-resistant antigen from M. tuberculosis, M. leprae and some other species of mycobacteria. Optimal binding results could be obtained within 4 h by the consecutive incubation of ML34-coated microtitre plates with antigen followed by the labelled ML34 antibody. The binding of intact bacilli was positive for M. tuberculosis but not for M. leprae. These results suggested that the MY4 antigen is expressed on the surface of M. tuberculosis and internally within M. leprae. Analysis of subcellular fractions suggested that this antigen is a constituent of cell walls.     

血清乙型肝炎病毒前S1抗原检测及其与病毒复制的关系

用抗Ｓ和抗前Ｓ１单抗的双抗体夹心ＥＬＩＳＡ法检测１５０例慢性乙型肝炎患者、乙型肝炎病毒表面抗原（ＨＢｓＡｇ）携带者和健康人血清中的ＨＢＶ前Ｓ１抗原，其结果和ＨＢＶＤＮＡ聚合酶链反应（ＰＣＲ）、乙型肝炎血清标志的检测结果进行比较。结果表明：前Ｓ１抗原在乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）阳性组中的检出率和相对滴度显著高于ＨＢｅＡｇ阴性组（Ｐ＜０．０１）；在ＨＢｅＡｇ阴性组中，抗－ＨＢｅ阴性人群前Ｓ１抗原的检出率和相对滴度也显著高于抗－ＨＢｅ阳性人群（Ｐ＜０．０１）。前Ｓ１抗原和ＨＢＶＤＮＡ检测结果的符合率达８０％，两者检出率的相关系数ｒ＝０．９８２６（Ｐ＜０．０１）。结论：血清前Ｓ１抗原和乙型肝炎病毒的存在关系密切。     

Sandwich type ELISA and a fluorescent cytometric microbead assay for quantitative determination of hepatitis B virus X antigen level in human sera

The hepatitis B virus X protein (HBxAg) is responsible for severe complications of HBV infections including primary hepatocellular carcinoma. A sandwich type ELISA and a flow cytometric microbead assay for quantitative determination of serum levels of Hbx-Ag are introduced. We have previously developed monoclonal antibody families against well-conserved epitopes on HbxAg, characterized by different immunohistochemical and immunoserological techniques. Special selection of the antibody pairs provided highly sensitive and highly specific tools for quantitative immunoassay development. The resulting assays were tested on human sera (208 samples) collected from patients suffering from different clinical forms of HBV infection. The sensitivity range of the sandwich type ELISA was between 4 and 2000 ng/ml as measured on both the recombinant antigen and the sera of chronic hepatitis patients. A further flow cytometric microbead assay was established and tested in parallel with the ELISA. The quantitative results of these two immunoserological techniques were in strong correlation and they were found to be highly specific and sensitive on clinical samples. The HBxAg ELISA technique is applicable for routine clinical laboratory measurements, and our HBxAg microbead technique is recommended for complex multiparametric measurements combined with other markers.     

Antibody detection and kinetics of antibody production during early stages of immunization with hepatitis B virus vaccine

Antibodies to influenza virus and human immunodeficiency virus are detectable in B cells during the early stages of the immune response, prior to their occurrence in plasma. To investigate similar phenomena in a model of immunization against hepatitis B virus (HBV) infection, medical students in Ghana were screened for HBV markers, HBV surface (HBs) antigen (HBsAg), and HBV core antibodies (anti-HBc). Consenting volunteers, 24 of whom were seronegative (susceptible) and 2 of whom were positive for anti-HBc (prior infection), were vaccinated on day 0, day 40, and 6 months. Two sets of 10 blood samples, sequentially collected at intervals of 2 days following each immunization on days 0 and 40, were processed into B-cell lysates and plasma. Solid-phase HBsAg coated on microtiter plates for enzyme immunoassay or nitrocellulose membranes for dot blot assay was used to detect anti-HBs activity by an indirect antiglobulin assay. A commercially procured sandwich immunoassay was used, along with an enzyme-linked immunosorbent assay and a dot blot assay, for the detection of anti-HBs in B-cell lysates and plasma. Following the first injection of vaccine, a single sample of B-cell lysate collected between 5 and 21 days revealed anti-HBs in 18/21 subjects with no plasma antibodies detectable by sandwich immunoassay. After the booster dose was injected on day 40, a single sample of B-cell lysate collected between 44 and 49 days showed anti-HBs in 16/19 subjects, and this was accompanied by plasma antibodies in 8 subjects. In contrast, between 8 and 13 days, both subjects with prior HBV infection showed anti-HBs in B-cell lysates and plasma. Thus, primary immunization with the HBV vaccine appears to transiently elicit low-affinity anti-HBs in B-cell lysates into plasma.     

Hepatitis B virus core-related antigens as markers for monitoring chronic hepatitis B infection

A sensitive chemiluminescence enzyme immunoassay has been developed for hepatitis B virus (HBV) core-related antigen (HBcrAg) detection. We aimed to investigate the usefulness of HBcrAg measurement for monitoring chronic hepatitis B disease. HBcrAg levels were measured by a chemiluminescence enzyme immunoassay in 54 untreated patients and 39 patients treated with either entecavir or lamivudine. The HBcrAg concentration correlated positively with the levels of serum HBV DNA (r = 0.820), intrahepatic total HBV DNA (r = 0.700), and covalently closed circular DNA (cccDNA) (r = 0.664; for all, P values were <0.001). A higher HBcrAg concentration was associated with a greater proportion of hepatitis B core antigen immunostaining. Although the differences were not statistically significant, patients with higher Knodell necroinflammation and fibrosis scores tended to have higher serum HBcrAg concentration levels. In the treated patients, the logarithmic reduction in HBcrAg at week 48 correlated positively with the logarithmic reduction of serum HBV DNA, intrahepatic total HBV DNA, and cccDNA. Of the 31 patients with undetectable serum HBV DNA (<300 copies/ml) at the end of treatment, 20 (65%) still had detectable HBcrAg. A greater reduction in posttreatment HBcrAg concentration was associated with histological improvement and a decrease in hepatitis B core antigen immunostaining. HBcrAg concentrations of <40,000 kU/ml at baseline and <200 kU/ml at week 24 were associated with a higher chance of having undetectable HBV DNA at week 48. In conclusion, serum HBcrAg levels correlated with HBV virological markers and reflected the chronic hepatitis B disease activity in the liver.     

Generation and characterization of a novel tetravalent bispecific antibody that binds to hepatitis B virus surface antigens

Hepatitis B virus (HBV) infection is a worldwide public health problem affecting about 350 million people. HBV envelope contains three surface antigens, called pre-S1, pre-S2 and S. For the prophylaxis of HBV infection, only an anti-S monoclonal antibody was tested for the protective efficacy against HBV infection, but it was shown to be incomplete. In addition, some immune escape mutants carrying mutations on the S antigen were reported. Therefore, a multivalent bispecific antibody rather than a single monoclonal antibody would be more beneficial for the prophylaxis of HBV infection. We have generated a novel tetravalent bispecific antibody with two binding sites for each of the S and pre-S2 antigens. Each of the antigen-binding sites was composed of a single-chain Fv (ScFv). The tetravalent antibody was generated by constructing a single gene encoding a single-chain protein. This protein consisted of an anti-S ScFv whose carboxyl end was tethered, through a 45 amino acid linker, to the amino terminus of anti-preS2 ScFv that in turn was joined to the hinge region of human γ1 constant region. The single-chain protein was expressed in Chinese hamster ovary cells and secreted in culture supernatant as a homodimeric molecule. The tetravalent bispecific antibody showed both anti-S and anti-pre-S2 binding activities. In addition, the binding affinity of the bispecific antiboy for HBV particles was greater than that of either parental antibody. The tetravalent bispecific antibody is a potentially useful reagent for the prevention and treatment of HBV infection.     

A point-of-care testing system with Love-wave sensor and immunogold staining enhancement for early detection of lung cancer

It has been reported that detection of exhaled breath condensate (EBC) is available for studies of pulmonary diseases, especially lung disease. In order to detect lung cancer (LC) at early stage, a point-of-care testing system suitable for measurement of tumor markers in EBC is developed. The assay, based on gold nanoparticle sandwich immunoassay and subsequent gold staining, was performed on a Love-wave sensor packaged inside a chip cartridge. Benefit from high sensitivity of Love-wave sensor, oriented immobilization of coating antibodies and immunogold staining enhancement, the present immunosensor could provide a sensitive, specific and rapid measurement. Carcinoembryonic antigen (CEA), neuron specific enolase (NSE) and squamous cell carcinoma antigen (SCC) in EBC collected from 17 patients with LC and 13 healthy volunteers were detected by this system. Results were compared with commercial chemiluminescence immunoassay and showed high correlation between two methods. Additionally, it revealed significantly statistical differences existing between two groups of subjects. These results indicate that the present system is suitable for detection of tumor markers in EBC and could be used as assistant tools for early detection of LC.     

A signal amplification system on a lateral flow immunoassay detecting for hepatitis e-antigen in human blood samples

Hepatitis B e-antigen (HBeAg) is the secretory form of the nucleocapsid of the hepatitis B virus (HBV), which is a marker of viral replication. In this study, a novel signal amplification system (SAS) based on the lateral flow immunoassay (LFIA) was used for rapid detection of HBeAg in blood samples from patients or blood donors. In this assay, the detection antibody was conjugated with gold nanoparticles (GNPs), and the capture antibody was labeled with biotin. The presence of targeting antigen HBeAg in blood sample would act as a bridge with biotinylated captured antibody and GNP-conjugated detection antibody to form the dendritic nanoparticle complex. The dendritic complexes in the sample solution were migrated and immobilized on the testing line of strip coated with antibiotin antibodies. Signal intensity was massively amplified by the SAS, which was positively correlated with the concentration of targeting antigen in the blood sample and was assessed by eyes or strip scanner. The SAS worked only when targeting antigens were present in the sample. By using this SAS-LFIA, we were able to detect a very low concentration of HBeAg (9 ng/mL), which was 27-fold sensitive than that by conventional LFIA (cLFIA). A number of 420 blood samples were detested by this novel SAS-LFIA, the results were in accordance with those of enzyme-linked immunosorbent assay (ELISA) completely, while the cLFIA missed an HBeAg-positive sample. In conclusion, the novel SAS has high specificity and sensitivity, which can be used to replace the conventional rapid test and ELISA in clinical diagnosis.     

Sensitive ELISA for IgG and IgM anti-albumin autoantibodies not influenced by HBsAg-associated polyalbumin receptors

An enzyme immunoassay for the detection of IgG and IgM anti-polymerized albumin autoantibodies (AAA) is described. It was found that polyalbumin receptors on HBsAg particles interfere in the detection of IgG AAA when polymerized human albumin (pHSA), but not polymerized bovine albumin (pBSA), is used as coating antigen. Polyalbumin receptors do not appear to interfere in the detection of IgM AAA, with either pHSA or pBSA as coating antigen. All normal sera showed evidence of AAA, of both IgG and IgM classes. Levels of IgG and IgM AAA in sera from most type A and type B acute hepatitis patients were above the range of normal controls. ELISA detection of AAA distinct from HBsAg reactivity can help in understanding the role of these autoantibodies in HBV infection.     

Levels of pre-S antigens and HBV DNA in sera from high and low viremic HBV carriers.

The biological and clinical significances of pre-S antigens and HBV replication were investigated. Some 125 sera, 28 from HBeAg and 97 from anti-HBe-positive HBsAg, carriers were studied. The aim was to verify whether pre-S antigens could be expressed in serum in complete absence of viremia. Pre-S proteins, determined by an enzyme immunoassay, were found in sera regardless of the presence of HBV DNA, as detected by spot-hybridization. The sera without detectable HBV DNA were investigated further by PCR using specific primers for the S and C regions of HBV. PCR analysis of samples revealed that 4 out of 5 HBeAg and 33 out of 41 (80.5%) anti-HBe positive sera contained HBV-amplified sequences of S and C regions. Pre-S antigen values correlated well with the amounts of HBV DNA in serum detected by PCR in anti-HBe-positive subjects with high titers of pre-S antigens (10(4)-10(6)). In addition, PCR highlighted the presence of HBV DNA sequences in 8 out of 17 (47.1%) pre-S-negative HBsAg-positive sera.     

Two vaccinia virus recombinants expressing HBsAg with different concentration of A- and pre-S2 antigenic determinants.

Comparative studies of two vaccinia virus (VV) recombinants expressing the hepatitis B virus (HBV) surface antigen (HBsAg) including the pre-S2 region (M-protein) showed that the L-pre-S2/15 recombinant expressed 5-fold more HBsAg as determined by the content of a-determinant than the recombinant v137. However, both recombinants expressed comparable amounts of the pre-S2 antigenic determinant as assessed by enzyme immunoassay with monoclonal antibodies. According to our calculations, one HBsAg unit expressed by the recombinant v137 contained 7-9 times more pre-S2 antigen than did one HBsAg unit expressed by the L-pre-S2/15 recombinant. Binding of pre-S2 region to polymerized human serum albumin was shown not to be an efficient assay at low pre-S2 concentration. HBsAg expressed by the v137 recombinant was less extensively secreted from cells as compared to that expressed by L-pre-S2/15 recombinant. Both recombinants induced the production of antibodies to the pre-S2 antigenic determinant in rabbits. L-pre-S2/15 induced anti-HBsAg a-determinant antibody as well.