Comparative immunogenicity of hepatitis B virus core and E antigens

The nucleocapsid (hepatitis B core Ag (HBcAg] of the hepatitis B virus is a particulate Ag composed of a single polypeptide (p21). Although a non-particulate form of HBcAg designated hepatitis B e Ag (HBeAg) shares significant amino acid identity, the immune responses to these Ag appear to be regulated independently. This report describes the use of recombinant HBcAg and HBeAg to examine and compare murine T cell and B cell recognition of these related Ag. The HBcAg preparation was stable at pH 7.2 and 9.6 and expressed HBc antigenicity. However, the antigenicity of the HBeAg preparation was pH dependent. At pH 9.6 the HBeAg preparation was non-particulate and expressed HBe antigenicity exclusively; however, at pH 7.2 it was particulate and expressed both HBc and HBe antigenicities. Although this "hybrid" particle most likely does not exist naturally, it is a unique research reagent to investigate the interrelationship between HBcAg and HBeAg. HBcAg was significantly more immunogenic in terms of in vivo antibody production as compared to either the non-particulate or particulate forms of HBeAg. Nevertheless, in most murine strains HBcAg and HBeAg were equivalently immunogenic and crossreactive at the level of T cell activation. The disparity between anti-HBc and anti-HBe antibody production is best explained by the observation that HBcAg can function as a T cell-independent Ag whereas HBeAg is T cell dependent even when present within the same particulate structure as HBcAg. Furthermore, HBcAg was shown to function efficiently as an immunologic carrier moiety for the DNP hapten in athymic as well as euthymic mice in contrast to conventional carrier proteins. These results have implications relevant to the human immune responses to HBcAg and HBeAg during infection, and to vaccine development.     

Identification of a major hepatitis B core antigen (HBcAg) determinant by using synthetic peptides and monoclonal antibodies

To map the location of hepatitis B core and e Ag (HBcAg and HBeAg) on the hepatitis B virus core particle, we produced and analyzed four synthetic peptides which correspond to the most hydrophilic regions of the core P22 protein. Each peptide was tested in an ELISA for the ability to inhibit the binding between rHBcAg or rHBeAg and either polyclonal or monoclonal anti-HBc or anti-HBe antibodies. The former comprised 20 antisera positive for anti-HBc (anti-HBs and anti-HBe negative) and five antisera positive for anti-HBe and anti-HBc; the latter included three anti-HBc mAb developed in independent laboratories: G6F5, C51B10, and F8, as well as two anti-HBe mAb, E2 and E6. These experiments revealed the presence of a major HBcAg epitope expressed on C3, a peptide which covers amino acids 107-118 and reacted with all polyclonal and monoclonal antibodies tested. Another peptide, C2, sequence 73-85, reacted with 26% of human antisera but none of the anti-HBc mAb. None of the peptides seemed to express HBeAg activity because they do not cause any significant inhibition of the HBeAg/anti-HBe reaction. These data indicate the expression of an immunodominant HBcAg determinant on a linear dodecapeptide and argue against a strict conformation dependency of this Ag.     

The hepatitis B virus core and e antigens elicit different Th cell subsets: antigen structure can affect Th cell phenotype.

Secretion of the hepatitis B virus (HBV) e antigen (HBeAg) has been conserved throughout the evolution of hepadnaviruses. However, the function of this secreted form of the viral nucleoprotein remains enigmatic. It has been suggested that HBeAg functions as an immunomodulator. We therefore examined the possibility that the two structural forms of the viral nucleoprotein, the particulate HBV core (HBcAg) and the nonparticulate HBeAg, may preferentially elicit different T helper (Th) cell subsets. For this purpose, mice were immunized with recombinant HBcAg and HBeAg in the presence and absence of adjuvants, and the immunoglobulin G (IgG) isotype profiles of anti-HBc and anti-HBe antibodies were determined. Second, in vitro cytokine production by HBcAg- and HBeAg-primed Th cells was measured. The immunogenicity of HBcAg, in contrast to that of HBeAg, did not require the use of adjuvants. Furthermore, HBcAg elicited primarily IgG2a and IgG2b anti-HBc antibodies, with a low level of IgG3, and no IgG1 anti-HBc antibodies. In contrast, the anti-HBe antibody response was dominated by the IgG1 isotype; low levels of IgG2a or IgG2b anti-HBe antibodies and no IgG3 anti-HBe antibodies were produced. Cytokine production by HBcAg- and HBeAg-primed Th cells was consistent with the IgG isotype profiles. HBcAg-primed Th cells efficiently produced interleukin-2 (IL-2) and gamma interferon (IFN-gamma) and low levels of IL-4. Conversely, efficient IL-4 production and lesser amounts of IFN-gamma were elicited by HBeAg immunization. The results indicate that HBcAg preferentially, but not exclusively, elicits Th1-like cells and that HBeAg preferentially, but not exclusively, elicits Th0 or Th2-like cells. Because HBcAg and the HBeAg are cross-reactive in terms of Th cell recognition, these findings demonstrate that Th cells with the same specificity can develop into different Th subsets based on the structural form of the immunogen. These results may have relevance to chronic HBV infection. Circulating HBeAg may downregulate antiviral clearance mechanisms by virtue of eliciting anti-inflammatory Th2-like cytokine production. Last, the influence of antigen structure on Th cell phenotype was not absolute and could be modulated by in vivo cytokine treatment. For example, IFN-alpha treatment inhibited HBeAg-specific Th2-mediated antibody production and altered the IgG anti-HBe isotype profile toward the Th1 phenotype.     

Immune response to hepatitis B virus core antigen (HBcAg): localization of T cell recognition sites within HBcAg/HBeAg

Hepatitis B virus nucleocapsid particles (HBcAg) can function as a T cell-independent antigen when injected into athymic mice. However, immunization of euthymic mice with HBcAg results in dramatically increased anti-HBc titers. Therefore we have examined the murine T cell response to HBcAg in terms of immunogenicity, the influence of H-2-linked genes, and the fine specificity of T cell recognition using synthetic peptide analogs. The HBcAg was shown to be an extremely efficient immunogen in terms of T cell activation as measured by the in vivo dose required to induce T cell sensitization (1.0 microgram), and the minimal in vitro concentration required to elicit interleukin 2 (IL 2) production (0.03 ng/ml). The degree of T cell immunogenicity of HBcAg and its ability to directly activate B cells most likely explain the enhanced humoral response to HBcAg in euthymic mice and HBV-infected patients. The influence of H-2-linked genes on the humoral response to HBcAg was discernable, and high responder (H-2k,s,d), intermediate responder (H-2b,f), and low responder (H-2p) haplotypes were identified. The H-2-linked regulation of the T cell response correlated with in vivo anti-HBc production. Examination of the fine specificity of T cell recognition revealed HBcAg-specific T cells from a variety of strains recognize multiple but distinct sites within the HBcAg/HBeAg sequence. T cell recognition sites were defined by small (16 to 21 residue) synthetic peptides. Each strain recognized a predominant T cell determinant, and the fine specificity of this recognition process was dependent on the H-2 haplotype of the responding strain. For example H-2s,b strains recognized p120-140, H-2f,q strains recognized p100-120, and H-2d mice recognized p85-100 predominantly. Because these sequences are common to both HBcAg and a nonparticulate form of the antigen termed HBeAg, these results indicate that HBcAg and HBeAg are highly cross-reactive at the T cell level although they are serologically distinct. These findings may have clinical relevance, because T cell sensitization to HBeAg and the subsequent seroconversion to anti-HBe status correlates with viral clearance during hepatitis B infection.     

Hepatitis B core antigen-specific IFN-gamma production of peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection

To evaluate the specificity of cellular immune response to hepatitis B virus (HBV) Ag in patients with chronic HBV infection, we have measured IFN-gamma production and proliferation of PBMC of 16 patients with chronic active hepatitis (CAH), 17 asymptomatic carriers of HBV (ASC), 6 anti-hepatitis B surface (HBs)-positive subjects, and 6 control individuals with ELISA procedure and [3H]thymidine incorporation. There was no significant increase in the mean proliferative response to recombinant HB surface and core Ag (rHBsAg and rHBcAg), nor was IFN-gamma production elicited with rHBsAg in any group. In contrast, PBMC of HBeAg-positive and anti-HBe-positive CAH patients, and anti-hepatitis B "e" Ag (HBe)-positive ASC showed significantly enhanced IFN-gamma production in response to HBcAg, whereas those of HBeAg-positive ASC and anti-HBs-positive subjects did not respond to HBcAg. The maximal response was observed in a 5-day culture with 500 ng/ml of rHBcAg when assessed by stimulation index value. Monocytes did not demonstrate an increased suppressor or helper activity for IFN-gamma production in these patients. T cell subset fractionation revealed that CD4+ cells were main population of IFN-gamma production specific for HBcAg and CD8+ cells did not suppress IFN-gamma production of CD4+ cells. Furthermore, CD4+ cells of HBeAg-positive ASC generated lesser amounts of IFN-gamma than HBeAg-positive CAH patients did. These results show that the measurement of IFN-gamma production is useful to determine cellular immune response to HBV Ag and suggest that IFN-gamma production depends on the helper activity of CD4+ T cells sensitized to HBcAg.     

乙型肝炎病毒e抗原的生物学功能及血清学转换的免疫学基础

目前在临床乙型肝炎的治疗中,乙型肝炎病毒e抗原(HBeAg)消失及其抗体的出现已成为重要的疗效指标.本文回顾了HBeAg的发现及其生物学和医学意义,对HBeAg与乙型肝炎病毒核心抗原(HBcAg)的免疫原性进行了比较,阐述了HBeAg血清学转换的免疫学基础,并对出现HBeAg血清学转换的意义作了分析.     

Activation of a heterogeneous hepatitis B (HB) core and e antigen-specific CD4+ T-cell population during seroconversion to anti-HBe and anti-HBs in hepatitis B virus infection.

Overcoming hepatitis B virus infection essentially depends on the appropriate immune response of the infected host. Among the hepatitis B virus antigens, the core (HBcAg) and e (HBeAg) proteins appear highly immunogenic and induce important lymphocyte effector functions. In order to investigate the importance of HBcAg/HBeAg-specific T lymphocytes in patients with acute and chronic hepatitis B and to identify immunodominant epitopes within the HBcAg/HBeAg, CD4+ T-cell responses to hepatitis B virus-encoded HBcAg and HBcAg/HBeAg-derived peptides were studied in 49 patients with acute and 39 patients with chronic hepatitis B. The results show a frequent antigen-specific CD4+ T-cell activation during acute hepatitis B infection, a rare HBcAg/HBeAg-specific CD4+ T-cell response among HBeAg+ chronic carriers, and no response in patients with anti-HBe+ chronic hepatitis. An increasing CD4+ T-cell response to HBcAg/HBeAg coincides with loss of HBeAg and hepatitis B virus surface antigen (HBsAg). Functional analysis of peptide-specific CD4+ T-cell clones revealed a heterogeneous population with respect to lymphokine production. Epitope mapping within the HBcAg/HBeAg peptide defined amino acids (aa) 1 to 25 and aa 61 to 85, irrespective of the HLA haplotype, as the predominant CD4+ T-cell recognition sites. Other important sequences could be identified in the amino-terminal part of the protein, aa 21 to 45, aa 41 to 65, and aa 81 to 105. The immunodominant epitopes are expressed in both proteins, HBcAg and HBeAg. Our findings lead to the conclusion that activation of CD4+ T lymphocytes by HBcAg/HBeAg is a prerequisite for viral elimination, and further studies have to focus on the question of how to enhance or induce this type of T-cell response in chronic carriers. The immunodominant viral sequences identified may have relevance to synthetic vaccine design and to the use of peptide T-cell sites as immunotherapeutic agents in chronic infection.     

Functional modulation of hepatitis B core antigen-specific T lymphocytes by an autoreactive T cell clone

Hepatitis B core (HBc)Ag-specific T cells present in the peripheral blood of a patient with chronic active hepatitis B were expanded by co-cultivation for 7 days with rHBcAg. After cloning at 1 cell/well in the presence of PHA and IL-2, five HBcAg-specific CD4+ cloned lines were obtained. All five lines proliferated and produced IL-2, IFN-gamma, and TNF in a dose-dependent fashion in response to HBcAg, but not to HBV envelope Ag. The cloned lines and derivative clones were HLA class II (DR1) restricted. All T cell clones were able to induce anti-HBc production by autologous B cells in response to HBcAg (helper effect). The proliferative response and the helper effect of the HBcAg-specific T cell lines and clones were augmented by co-cultivation with an autologous, autoreactive (HLA-DQ1 specific) T cell clone, even in the absence of HBcAg, and the autoreactive T cells directly stimulated anti-HBc secretion by autologous B cells, presumably due to the release of Ag-nonspecific factors. These findings define a model immunoregulatory circuit the physiologic significance of which remains to be determined.     

Functional characterization of cloned intrahepatic, hepatitis B virus nucleoprotein-specific helper T cell lines

We have previously reported the establishment and preliminary characterization of polyclonal hepatitis B virus (HBV) nucleoprotein (HBcAg)-specific CD4+ and CD8+ T cell lines derived from the hepatic lymphomononuclear cell infiltrate of several patients with chronic active hepatitis B. The isolated subsets from these lines were specifically activated by HBcAg and displayed antigen-specific help and suppression with respect to proliferation of the alternate subset. One of these lines was recently cloned by limiting dilution, and four HBcAg-specific CD3+ CD4+ CD8-DR+ T cell lines were produced that had a 95.3% likelihood of monoclonality. Antigenic specificity was confirmed by dose-dependent, HLA class II (DR)-restricted proliferation in response to recombinant and human serum-derived HBcAg and the lack of proliferation to HBV envelope antigens (HBsAg and pre-S(2)Ag). All cloned lines were interleukin 2 dependent, produced interferon-gamma in an antigen-specific manner, and provided antigen-specific help to autologous B cells with respect to anti-HBc production. We conclude that HBcAg-specific, HLA-class II restricted helper T cells capable of inducing antigen-specific functional responses by autologous B lymphocytes and T lymphocytes are present at the site of viral antigen synthesis and hepatocellular injury in HBV infection.     

Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5-20 ng RNA/microg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-gamma release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20-100 microg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 microg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 microg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.     

Prevalence of hepatitis B viral markers in Vietnamese blood donors

Serum samples were assayed using radioimmunoassay in 573 Vietnamese blood donors living in Hano? (North Viet Nam). 66 (11.5%) subjects were HBsAg-positive. Of these 66 HBsAg carriers, 17 (25,8%) were positive for hepatitis B e antigen (HBeAg) and 43 (65.1%) for antibody to HBeAg (anti-HBe). 22 (3.8%) subjects were positive for antibody to hepatitis B core antigen (anti-HBc) alone. 402 (70.2%) subjects were positive for antibody to HBsAg (anti-HBs). This anti-HBs percentage increased with age. Only 83 (14.5%) subjects were negative for all hepatitis B viral (HBV) markers. This no HBV markers percentage decreased with age. The chi 2 test showed a non significant difference for frequencies of HBsAg, anti-HBc alone, anti-HBs but a significant one for frequencies of no HBV markers in men and women.     

Age-specific prevalence of hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe) among asymptomatic hepatitis B surface antigen (HBsAg) carriers in Okinawa and Kyushu, Japan

A total of 1,741 asymptomatic hepatitis B surface antigen (HBsAg) carriers in two areas (Okinawa and Kyushu) in Japan were surveyed for the presence of hepatitis B e antigen (HBeAg) and the corresponding antibody (anti-HBe) to determine the age-specific prevalence of these markers and the mean age of carriers with HBeAg. Prevalence of HBeAg was significantly higher in Kyushu (36.4% of 755 carriers) than in Okinawa (20.0% of 986 carriers) (P less than 0.001). The mean age of carriers with HBeAg was 25.5 years in Kyushu and 16.1 years in Okinawa, suggesting that HBeAg converted to anti-HBe earlier in Okinawa than in Kyushu. In contrast, the prevalence of anti-HBe was significantly higher in Okinawa (74.6% of 986) than in Kyushu (56.3% of 755) (P less than 0.001). The prevalence of HBeAg decreased with age up to 40-49 years of age and then increased in both areas. Prevalence of anti-HBe was inversely related to the prevalence of HBeAg in both areas. These data suggest that HBeAg and anti-HBe are chronological markers of chronic hepatitis B virus infection and that the duration of HBeAg persistence can be different in different area, even in the same country.     

Intrahepatic, nucleocapsid antigen-specific T cells in chronic active hepatitis B

Hepatitis B core antigen (HBcAg)-specific T cell lines were established from hepatic lymphomononuclear cells derived from five patients with chronic active hepatitis B. No hepatitis B virus envelope antigen-specific cell lines were established. Proliferation in response to recombinant and native HBcAg, but not to native hepatitis B surface antigen containing the pre-S(2) region, confirmed the specificity of the five T cell lines. All cell lines represented mixed populations of CD4+ and CD8+ T cells. The CD4+ subset provided antigen-specific help to autologous B cells with respect to anti-HBc production and to CD8+ cells with regard to HBcAg-induced proliferation and suppressor activity. The CD8+ subset contained suppressor cells that selectively inhibited the proliferative response of autologous HBcAg-specific CD4+ cells without inhibiting CD4+ cells of unrelated specificity (tetanus toxoid). Moreover, the CD8+ cells were also capable of suppressing HBcAg-stimulated antibody to HBcAg production without showing inhibition of total immunoglobulin production stimulated by pokeweed mitogen. The cytotoxic potential of the T cell lines was established in a lectin-dependent cytotoxicity system; natural killer cytotoxicity was completely absent. Our data suggest that the lesional T cells present at the site of hepatocellular injury in chronic active hepatitis B are primarily HBcAg-specific lymphocytes of the helper and suppressor/cytotoxic phenotypes and that both are functionally competent.     

Modulation of hepatitis B infection by intravenous application of an immunoglobulin preparation that contains antibodies to hepatitis B e and core antigens but not to hepatitis B surface antigen.

Repeated administration of an intravenous immunoglobulin containing antibody to hepatitis B e antigen (anti-HBe) and antibody to hepatitis B core antigen (anti-HBc) but free of antibody to hepatitis B surface antigen (anti-HBs) before and after the inoculation of 10(4.9) 50% chimpanzee infective doses of hepatitis B virus (HBV) markedly prolonged the incubation period of HBV in experimentally infected chimpanzees. Similar administration of an immunoglobulin preparation containing anti-HBc but free of anti-HBe and anti-HBs or intramuscular administration of a single dose of immunoglobulin containing anti-HBe and anti-HBc 3 days before or after inoculation with HBV did not appear to modulate HBV infection. These observations suggested that anti-HBe, or an unidentified antibody associated with it, may have biological activity in the modulation of HBV replication.     

HBcAg-specific CD4+CD25+regulatory T cells modulate immune tolerance and acute exacerbation on the natural history of chronic hepatitis B virus infection

Acute exacerbations (AEs) of chronic hepatitis B (CH-B) are accompanied by increased T cell responses to hepatitis B core and e antigens (HBcAg/HBeAg). Why patients are immunotolerant (IT) to the virus and why AEs occur spontaneously on the immunoactive phase remain unclear. The role of HBcAg-specific CD4(+)CD25(+) regulatory T (T(reg)) cells in AE and IT phases was investigated in this study. The SYFPEITHI scoring system was employed to predict MHC class II-restricted epitope peptides on HBcAg overlapping with HBeAg that were used for T(reg)-cell cloning and for the construction of MHC class II tetramers to measure T(reg) cell frequencies (T(reg) f). The results showed that HBcAg-specific T(reg) f declined during AE accompanied by increased HBcAg peptide-specific cytotoxic T lymphocyte frequencies. Predominant Foxp3-expressing T(reg) cell clones were generated from patients on the immune tolerance phase, while the majority of Th1 clones were obtained from patients on the immunoactive phase. T(reg) cells from liver and peripheral blood of CH-B patients express CD152 and PD1 antigens that exhibit suppression on PBMCs proliferation to HBcAg. These data suggest that HBcAg peptide-specific T(reg) cells modulate the IT phase, and that their decline may account for the spontaneous AEs on the natural history of chronic hepatitis B virus infection.     

Peripheral T-cell subsets in chronic type B hepatitis: correlation with biochemical and histological activities and hepatitis B e antigen/antibody status

Peripheral T-cell subsets in 77 patients with hepatitis B surface antigen (HBsAg)-positive chronic liver diseases were studied by indirect immunofluorescence using murine monoclonal antibodies against all peripheral T cells (OKT3), T-helper/inducer cells (OKT4), and T-cytoxic/suppressor cells (OKT8). OKT4/OKT8 ratios were significantly reduced in patients with hepatitis B e antigen (HBeAg)-positive chronic liver diseases, including 28 patients with chronic active hepatitis (CAH) (P less than 0.001) and 15 with chronic persistent hepatitis (CPH) (P less than 0.001). OKT4/OKT8 ratios were significantly lower in 21 HBeAg-negative patients with CAH (P less than 0.05), as compared to those of 17 normal controls, while T-cell subsets in 13 patients with HBeAg-negative CPH were essentially normal. Low OKT4/OKT8 ratios significantly correlated with HBeAg positivity (P less than 0.001) and CAH (P less than 0.05), as assessed with multiple regression. There was a significant negative correlation between OKT4/OKT8 ratios and serum glutamic-pyruvic transaminase (SGPT) levels (r = -0.37; P less than 0.01). It was concluded that in chronic hepatitis B virus infection, low OKT4/OKT8 ratios are closely related to active viral replication and more severe histological and biochemical activity.     

Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection

The proliferative response of PBMC to hepatitis B virus (HBV) envelope, core, and e Ag was analyzed prospectively in 21 patients with acute self-limited HBV infection and compared with the response of patients with chronic HBV infection and different levels of HBV replication (i.e., hepatitis e Ag (HBeAg)- or anti-HBe-positive) and liver damage (i.e., chronic active hepatitis or chronic asymptomatic carriers). Our results indicate that: 1) HBV-infected subjects who develop a self-limited acute hepatitis show a vigorous PBMC response to hepatitis B core Ag and HBeAg, as expression of T cell activation; 2) appearance of a detectable lymphocyte response to HBV nucleocapsid Ag is temporally associated with the clearance of HBV envelope Ag; 3) in patients with chronic HBV infection the level of T cell responsiveness to hepatitis B core Ag and to HBeAg is significantly lower than that observed during acute infection; 4) T cell sensitization to HBV envelope Ag in acute and chronic HBV infection is usually undetectable and when measurable is expressed transiently and at low levels. These results may reflect immune events of pathogenetic relevance with respect to evolution of disease and viral clearance.     

Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies

Mice were immunized against hepatitis B e antigen (HBeAg) isolated from sera of asymptomatic carriers of hepatitis B virus. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 5 clones of hybridoma cells secreting antibody against HBeAg (anti-HBe) were isolated. For the production of anti-HBe in large scale, cells were cultivated both in vitro and in the peritoneal cavity of ascitic mice. Although monoclonal antibodies produced by these clones showed a strong reactivity of anti-HBe in hemagglutination tests, individual monoclonal anti-HBe did not reveal any precipitin line in immunodiffusion. When 2 of the 5 monoclonal antibodies were mixed together, however, some combinations showed a precipitin line against HBeAg, whereas others did not. Utilizing solid-phase radioimmunoassay involving a number of combinations of monoclonal antibodies used for solid-phase and radiolabeling, the 5 antibodies were classified into 2 groups. Three of the anti-HBe antibodies were found to be directed to 1 determinant of HBeAg (determinant a); the remaining 2 to the other determinant (determinant b). Determinants a and b were detected on HBeAg in the serum, as well as on the polypeptide of 19,000 daltons (P19) derived from the nucleocapsid of hepatitis B virus. Monoclonal anti-HBe antibodies with different specificities may provide useful tools in delineating the antigenic structure of HBeAg and also in evaluating immune responses of the host directed to its subdeterminants.     

Suitability of yeast- and Escherichia coli-expressed hepatitis B virus core antigen derivatives for detection of anti-HBc antibodies in human sera

Antibody to hepatitis B virus core antigen (anti-HBc) is one of the most important serological markers during hepatitis B virus (HBV) infection. The quality of the hepatitis B virus core antigen (HBcAg; diagnostic antigen) is crucial to the accuracy of anti-HBc detection. In an attempt to explore the suitability of recombinant HBcAg (rHBcAg) for diagnostic purposes, HBcAg was expressed in Escherichia coli (E. coli) and Pichia pastoris (P. pastoris) and evaluated for the detection of anti-HBc. The expression level of the recombinant protein satisfied the criteria for large-scale biologic production. P. pastoris- and E. coli-derived rHBcAg were purified with gel filtration followed by sucrose gradient (reagents A and C) or with a monoclonal anti-HBc antibody binding (reagents B and D) and were utilized to detect anti-HBc in competitive inhibition enzyme-linked immunosorbent assay (ELISA) format. The ELISA using P. pastoris-derived rHBcAg had a higher specificity and sensitivity than that using E.coli-derived rHBcAg to detect the anti-HBc standard panel. Serum specimens were collected from HBV-infected patients and healthy individuals (voluntary blood donors). Anti-HBc was detected in those specimens using P. pastoris- and E. coli-derived rHBcAg. The positive rate of anti-HBc detection in HBV-infected patients' sera was 100% with reagents A and B, 96.4% with reagent C, and 93.6% with reagent D. The negative rate in healthy control sera was 100% with reagents A and B, 97.0% with reagent C, and 99.7% with reagent D. These data indicate that P. pastoris-derived rHBcAg is superior to E.coli-derived rHBcAg for the detection of anti-HBc using the diagnostic ELISA.     

大肠杆菌合成的HBcAg转化为HBeAg的研究及其应用

37℃用1％β-巯基乙醇处理大肠杆菌HBcAg2小时,可使其全部转化成HBeAg。以常规ELISA方法不能检出HBcAg活性。该制品能被抗-HBe阳性血清特异中和。4℃以液体形式存放稳定,-20℃经过冻融活性下降。HBcAg转化成HBeAg后聚丙烯酰胺凝胶电泳行为有所改变。分别用转化的大肠杆菌HBeAg和血浆HBeAg作为酶联反应的诊断试剂,检测35份HBsAg阳性乙肝病人血清标本的抗-HBe,符合率为91.4％。同用Abbott-HBe(RIA)试剂盒测定的17份HBsAg阳性血清标本的试验结果比较,符合率为100％。说明转化的大肠杆菌HBeAg可代替血浆HBeAg作为检测抗-HBe的诊断试剂应用。