Clinical and Virological Characteristics of Chronic Hepatitis B Patients with Coexistence of HBsAg and Anti-HBs

Coexistence of hepatitis B surface antigen (HBsAg) and antibody against HBsAg (anti-HBs) comprises an atypical serological profile in patients with chronic hepatitis B virus (HBV) infection. In this study, in total 94 patients with coexisting HBsAg and anti-HBs and 94 age- and sex-matched patients with positive HBsAg were characterized by quantitatively measuring HBsAg and HBV DNA, sequencing large S genes, and observing clinical features. Compared with common hepatitis B patients, the patients with coexisting HBsAg and anti-HBs had lower HBsAg and HBV DNA levels. These two groups had similar rate of pre-S deletion mutations. However, in patients with coexisting HBsAg and anti-HBs, more amino acid substitutions in the a determinant of S gene were observed in HBV genotype C, but not in genotype B. Fourteen patients with coexisting HBsAg and anti-HBs were followed up for an average of 15.5 months. There were no significant changes in the levels of HBsAg, anti-HBs, HBV DNA and ALT over the follow-up period. Compared with the baseline sequences, amino acid substitutions in the MHR of HBsAg occurred in 14.3% (2/14) patients. In conclusion, coexistence of HBsAg and anti-HBs may be associated with higher frequency of mutations in the a determinant of HBV genotype C.     

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.     

Algorithm of serologic screening and assessment of prevalence of serologically meaningful mutations of HBsAg in hepatitis B virus carriers

Algorithm of serologic screening for HBsAg-mutants in hepatitis B virus (HBV) carriers with high level of HBsAg was developed which is based on the detection of defects of interactions of serum HBsAg with monoclonal anti-HBs realizing as a decrease of ELISA sensitivity in 10 times or more during serial 10-fold dilutions. During 1st stage commercial test-systems based on monoclonal antibodies was used to select serum samples with discrepancy of test results. During 2nd stage HBsAg contained in selected sera was analyzed by the panel of monoclonal and polyclonal anti-HBs conjugates using decrease in ELISA sensitivity as a criterion. Serum samples from 2510 chronic carriers of HBV with high level of HBsAg were studied. 19 samples with discrepant results were found. Subsequent characterization of HBsAg with panel of 11 monoclonal and 1 polyclonal conjugates allowed to distinguish groups of sera with specific serologic "portraits". Atypical features of HBsAg were confirmed by genotyping 9 of 19 samples. Analysis of primary nucleotide sequence revealed serologically meaningful mutations in S-gene of HBV in all 9 isolates: 3 of them contained substitution mutation G145R, 5--S143L, and one--T143M. Distribution of mutations in HBsAg corresponded with specific serologic "portraits". Prevalence of HBsAg mutations in HBV carriers with high level of HBsAg was assessed for the first time: prevalence of G145R, S143L/T143M mutations, and all serologically atypical variants was 0.12%, 0.24%, and 0.76% respectively. Developed algorithm was proposed for epidemiologic monitoring of HBsAg-mutants of HBVand control of diagnostic test-systems.     

G145R突变后乙型肝炎病毒表面抗原在酵母系统的表达及其免疫学特性分析

为了研究乙型肝炎(乙肝)病毒表面抗原(HBsAg)发生G145R突变后的免疫学特性改变情况,首先利用Pichia pastoris酵母表达系统分泌表达G145R突变后的HBsAg的preS2 S(中蛋白),用重组表达产物免疫小鼠,酶联免疫吸附试验(ELISA)和Western blot实验等研究其抗原性和免疫原性与野毒型HBsAg的异同。从150个阳性表达克隆中筛选出一株表达量最高的克隆株MC23,Western blot检测显示,表达的HBsAg中蛋白单体主带分子量在34kD、37kD左右,表达量约为200μg／L。用不同的HBsAg检测试剂检测其抗原性发现,G145R突变后的HBsAg,用绝大多数试剂都不能很好地检出,检出能力只有野毒型HBsAg的50％或更低,但用美国雅培公司的试剂检出能力可达野毒的98％。G145R突变后的HBsAg中蛋白免疫小鼠后,血清中可检测到1：1600的特异性表面抗体,该抗体与G145R突变后的HBsAg“a”决定簇合成肽P2—145R也能发生交叉反应,反应滴度为1：80。但该抗体和野毒型HBsAg蛋白以及野毒“a”决定簇合成肽P1-wt均不反应。上述结果表明,G145R突变后的HBsAg中蛋白在Pichia pastoris酵母系统得到了分泌表达,表达产物仍具有较好的免疫原性,但和野毒HBsAg相比,其抗原性和免疫原性发生了明显改变。     

Endocytosis of hepatitis B immune globulin into hepatocytes inhibits the secretion of hepatitis B virus surface antigen and virions

Hepatitis B immunoglobulin is used for prophylaxis against hepatitis B virus (HBV) and is thought to act by neutralization of virions and hepatitis B virus surface antigen (HBsAg)-containing particles in circulation. Using a panel of hepatocyte-derived cell lines, the present study investigated in vitro whether HBs-specific immunoglobulin G (IgG) is internalized in hepatocytes and whether it interacts with HBsAg in the cells. By immunoelectron microscopy and immunoblotting, human IgG and FcRn receptor for IgG were demonstrated on cellular membranes and in cytoplasmic extracts, irrespective of the HBsAg status of the cells. Furthermore, HBsAg and anti-HBs were shown to be colocalized in the same cellular compartment by two-color confocal microscopy. Endocytosis of HBs-specific IgG caused intracellular accumulation of HBsAg in a dose-dependent manner and inhibited the secretion of HBsAg and HBV virions from the cells. These effects were not observed with F(ab)(2) fragments or nonimmune IgG as controls. The specificity of intracellular HBsAg- anti-HBs interaction was further investigated in cells transfected with HBV genomes expressing wild-type HBsAg or immune escape HBsAg (with a G145R mutation). Monoclonal anti-HBs markedly reduced the secretion of wild-type HBsAg, while the secretion of mutant HBsAg was not affected. These results suggest that HBs-specific IgG binds to hepatocytes and interacts with HBsAg within the cells. This may be relevant for the selection of surface antibody escape mutations.     

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.     

重组质粒与重组蛋白共免疫诱导HBsAg特异性T细胞免疫抑制

摘要：【目的】为了探索治疗急性乙型肝炎和爆发性乙型肝的新策略,本研究将HBV DNA疫苗和相应抗原的蛋白质分子联合免疫小鼠,旨在探讨联合免疫对小鼠抗原特异性T细胞增殖反应的影响。【方法】我们将HBV DNA疫苗（pcDS2）和相应抗原蛋白质分子(HBsAg)联合免疫BALB/c小鼠;分别在第0、2和4周进行免疫,在第6周用ELISA方法检测抗-HBs IgG效价,MTT和流式细胞仪检测T细胞增殖反应,及流式细胞仪检测细胞因子表达水平。【结果】pcDS2和HBsAg联合免疫组小鼠的抗-HBs水平显著提高;免疫小鼠的T细胞体外经HBsAg刺激后, 联合免疫组刺激指数(SI)明显降低;经流式细胞仪检测进一步证实联合免疫组T细胞增殖反应被显著抑制;联合免疫组T细胞表达IL-10和Foxp3水平显著升高。【结论】pcDS2和HBsAg联合免疫能诱导产生特异性体液免疫应答,但不能诱导产生抗原特异性T细胞增殖反应;T细胞增殖反应被显著抑制可能与T细胞表达IL-10和Foxp3上调有关;本研究为急性乙型肝炎和爆发性乙型肝炎治疗及HBV疫苗的研究奠定了基础。     

重庆地区乙肝血清特殊模式患者HBV基因分型及临床自然病程分析

目的了解重庆地区乙肝病毒（HBV）血清学标志物为特殊模式的HBV感染患者病毒基因型的分布情况,分析其临床特征及自然病程。方法从1000例HBV感染者中检测到48例乙肝病毒血清学标志物为特殊模式的患者（HBsAg与抗一HBs同时阳性,HBeAg与抗一HBe同时阳性）。采用巢式聚合酶链式反应（nPCR）对特殊模式患者的HBV进行基因分型,同时对两组特殊模式患者的临床资料和HBV感染的自然史进行分析。结果48例乙肝病毒血清学标志物为特殊模式的HBV感染者中,36例患者HBsAg与抗-HBs同时阳性,12例患者HBeAg与抗-HBe同时阳性。HBeAg＋／抗-HBe＋患者组的年龄较HBsAg＋／抗-HBs＋患者组的小（P〈0．05）。HBsAg＋／抗-HBs＋患者中,3例（8．3％）为B2亚型,12例（33．3％）为c2亚型,21例（58．4％）未分型;HBeAg＋／抗-HBe＋患者中,8例（66．7％）为B2亚型,1例（8．3％）为c2亚型,3例（25．0％）未分型,两组在HBV基因型的分布上差异具有统计学意义（Y2=17．44,P〈0．05）。在HBsAg＋／抗-HBs＋患者中,2例（4．2％）处于免疫清除期,14例（29．2％）处于低复制期,7例（14．6％）处于再活动期。HBeAg＋／抗-HBe＋患者中,5例（10．4％）处于免疫清除期。两组在HBV感染的自然病程中的分布差异具有统计学意义（X2=18．26,P〈0．05）。结论重庆地区乙肝病毒血清学标志物为特殊模式的慢性HBV感染者中,HBeAg与抗-HBe同时阳性的HBV感染者中B2亚型为优势基因型;HBsAg与抗-HBs同时阳性的HBV感染者中,HBV基因型以C2亚型为主。     

Gly145 to Arg substitution in HBs antigen of immune escape mutant of hepatitis B virus.

A Japanese child born to an HBeAg-positive carrier mother received anti-HBs immunoglobulins and a plasma-derived HBs vaccine with a poor anti-HBs-antibody response. The child, who is now 3 years old, is presently suffering from chronic hepatitis with unusual serological findings that are positive for HBsAg, anti-HBs and HBeAg, since being infected with a measles virus at 12 months of age. The nucleotide sequences of the S region of HBV DNA obtained from the patient, the mother and an HBeAg-positive brother were completely identical except for one nucleotide at position 587 (mother and brother: guanosine, patient: adenosine), giving an amino acid change: Gly - greater than Arg at position 145 of the major HBs protein.     

DNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV) core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic

Background

Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4) primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV) clearance.

Principal Findings

Plasmids expressing HBV core protein (HBcAg) or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc), CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI) of pAAV/HBV1.2. HBV surface antigen (HBsAg) and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance.

Conclusion

Viral clearance could be efficiently achieved by Th1/Th2-balanced immune response, with a small but significant shift in T-cell and B-cell immune responses.     

Impairment of hepatitis B virus virion secretion by single-amino-acid substitutions in the small envelope protein and rescue by a novel glycosylation site

Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site ((131)NST(133)). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.     

Investigation of Occult Hepatitis B Virus Infection in Anti-HBc Positive Patients from a Liver Clinic

Occult hepatitis B infection (OBI) is manifested by presence of very low levels (<200IU/mL) of Hepatitis B viral DNA (HBV DNA) in the blood and the liver while exhibiting undetectable HBV surface antigen (HBsAg). The molecular mechanisms underlying this occurrence are still not completely understood. This study investigated the prevalence of OBI in a high-risk Australian population and compared the HBV S gene sequences of our cohort with reference sequences. Serum from HBV DNA positive, HBsAg negative, and hepatitis B core antibody (anti-HBc) positive patients (study cohort) were obtained from samples tested at SEALS Serology Laboratory using the Abbott Architect, as part of screening and diagnostic testing. From a total of 228,108 samples reviewed, 1,451 patients were tested for all three OBI markers. Only 10 patients (0.69%) out of the 1,451 patients were found to fit the selection criteria for OBI. Sequence analysis of the HBV S gene from 5 suspected OBI infected patients showed increased sequence variability in the ‘a’ epitope of the major hydrophilic region compared to reference sequences. In addition, a total of eight consistent nucleotide substitutions resulting in seven amino acid changes were observed, and three patients had truncated S gene sequence. These mutations appeared to be stable and may result in alterations in HBsAg conformation. These may negatively impact the affinity of hepatitis B surface antibody (anti-HBs) and may explain the false negative results in serological HBV diagnosis. These changes may also enable the virus to persist in the liver by evading immune surveillance. Further studies on a bigger cohort are required to determine whether these amino acid variations have been acquired in the process of immune escape and serve as markers of OBI.     

A case of hepatitis B reactivation in an anti-HBs positive, anti-HBc positive non-Hodgkin’s lymphoma patient

Dear Editor, We report a case of HBV reactivation in an anti-HBs positive,anti-HBc positive non-Hodgkin's lymphoma patient.Hepatitis B virus (HBV) reactivation is a well-recognized complication of patients undergoing chemotherapy or immunosuppressive therapy for lymphomas.The presence of antibodies to the hepatitis B surface antigen (anti-HBs) has been identified to be a factor preventing HBV reactivation in patients with occult HBV infection receiving chemotherapy.In this paper,we present a non-Hodgkin Lymphoma patient who,before immunosuppressive therapy,displayed positive anti-HBs and positive antibodies to hepatitis B core antigens (anti-HBc),as markers of resolved HBV infection,and developed hepatitis B surface antigen (HBsAg) and high viraemia with an HBV escape mutant after rituximabbased administration.The sequencing data revealed HBV genotype D with two known escape mutations,P120S.     

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.     

Evaluation of the G145R Mutant of the Hepatitis B Virus as a Minor Strain in Mother-to-Child Transmission

The role of the hepatitis B virus (HBV) mutant G145R, with a single change in amino acid 145 of the surface protein, as a minor population remains unknown in mother-to-child transmission. The minor strain as well as the major strain of the G145R mutant were evaluated in three cohorts using a locked nucleic acid probe-based real-time PCR. The breakthrough cohort consisted of children who were born to HBV carrier mothers and became HBV carriers despite immnoprophylaxis (n = 25). The control cohort consisted of HBV carriers who had no history of receiving the hepatitis B vaccine, hepatitis B immunoglobulin or antiviral treatment (n = 126). The pregnant cohort comprised pregnant women with chronic HBV infection (n = 31). In the breakthrough cohort, 6 showed positive PCR results (major, 2; minor, 4). In the control cohort, 13 showed positive PCR results (major, 0; minor, 13). HBeAg-positive patients were prone to have the G145R mutant as a minor population. Deep sequencing was performed in a total of 32 children (PCR positive, n = 13; negative, n = 19). In the breakthrough cohort, the frequency of the G145R mutant ranged from 0.54% to 6.58%. In the control cohort, the frequency of the G145R mutant ranged from 0.42% to 4.1%. Of the 31 pregnant women, 4 showed positive PCR results (major, n = 0; minor, n = 4). All of the pregnant women were positive for HBeAg and showed a high viral load. Three babies born to 3 pregnant women with the G145R mutant were evaluated. After the completion of immunoprophylaxis, 2 infants became negative for HBsAg. The remaining infant became negative for HBsAg after the first dose of HB vaccine. G145R was detected in one-fourth of the children with immunoprophylaxis failure. However, the pre-existence of the G145R mutant as a minor population in pregnant women does not always cause breakthrough infection in infants.     

Modulation of hepatitis B virus secretion by naturally occurring mutations in the S gene

Alteration in hepatitis B virus (HBV) secretion efficiency may have pathological consequences. Naturally occurring mutations that regulate virion secretion have not been defined. We recently identified HBV genomes displaying high (4B), substantially reduced (3.4), or negative (4C) virion secretion. In the present study, the underlying mutations were mapped. A T552C point mutation in the 4B genome was responsible for its enhanced virion secretion, whereas a G510A mutation in 3.4 and G660C in 4C impaired virus secretion. The three point mutations generate M133T, G119E, and R169P substitutions in the S domains of viral envelope proteins, respectively, without modifying the coding capacity of the overlapping polymerase gene. The mutated residues are predicted to lie in the luminal side of the endoplasmic reticulum (ER) or to be embedded in the ER membrane and thus are not involved in contact with core particles during envelopment. Of the two mutations inhibitory of virion secretion, G510A greatly reduced small envelope protein (hepatitis B surface antigen [HBsAg]) levels both inside cells and in culture medium, whereas G660C specifically abolished HBsAg secretion. Surprisingly, a T484G mutation in the 4B genome, generating an I110M substitution in the S domain, could also reduce HBsAg secretion and block virion secretion. However, its inhibitory effect was suppressed in the 4B genome by the T552C mutation, the enhancer of virion secretion. T552C can also override the inhibitory G510A mutation, but not the G660C mutation. These findings suggest a hierarchy in the regulation of virion secretion and a close link between defective virion secretion and impaired HBsAg formation or secretion.     

Variability of the hepatitis B surface protein in HBV-infected liver transplant recipients

Variations in the major surface proteins (HBsAg) of hepatitis B virus (HBV) have been implicated in the high rate of reinfection in HBV-infected recipients of orthotopic liver transplantations (OLT). Sera from 6 OLT patients positive for HBsAg and from 3 recipients negative for it prior to transplantation were analyzed over several years, and 39 HBsAg sequences were compared. Despite anti-HBs immunoprophylaxis resulting in the disappearance of HBsAg, HBV DNA was detectable by a sensitive nested PCR in almost all sera. In 1 patient, a significant temporary shift in HBV subtypes was observed, indicating a mixed infection or the presence of multiple HBV populations in this patient; this was also true for other patients. Amino acid substitutions compared to wild-type HBV subtypes in 7 patients and variations within patients in 5 patients were detectable over time; the escape mutation at amino acid position 145 was detected in 2 patients. Our data suggest that the high rate of reinfection in OLT recipients seems not to be associated with specific sequence variations in the major HBs gene, but shows a remarkable inter- and intraindividual variability. Obviously, no correlation between heterogeneity in this gene and clinical outcome was present.The following investigators and institutions were members of the Liver Transplantation Group: U. Beuers, M. Bilzer, W. Caselmann, A. Gerbes, R. Hoffmann, C. Jung, G.R. Pape (Medical Department II), J. Briegel, J. Groh, M. Haller (Institute of Anesthesiology), H.J. Krämling, H. Rauh and M. Stangl (Department of Surgery).     

Persistence of the Recombinant Genomes of Woodchuck Hepatitis Virus in the Mouse Model

Hydrodynamic injection (HI) with a replication competent hepatitis B virus (HBV) genome may lead to transient or prolonged HBV replication in mice. However, the prolonged HBV persistence after HI depends on the specific backbone of the vector carrying HBV genome and the genetic background of the mouse strain. We asked whether a genetically closely related hepadnavirus, woodchuck hepatitis virus (WHV), may maintain the gene expression and replication in the mouse liver after HI. Interestingly, we found that HI of pBS-WHV1.3 containing a 1.3 fold overlength WHV genome in BALB/c mouse led to the long presence of WHV DNA and WHV proteins expression in the mouse liver. Thus, we asked whether WHV genome carrying foreign DNA sequences could maintain the long term gene expression and persistence. For this purpose, the coding region of HBV surface antigen (HBsAg) was inserted into the WHV genome to replace the corresponding region. Three recombinant WHV-HBV genomes were constructed with the replacement with HBsAg a-determinant, major HBsAg, and middle HBsAg. Serum HBsAg, viral DNA, hepatic WHV protein expression, and viral replication intermediates were detected in mice after HI with recombinant genomes. Similarly, the recombinant genomes could persist for a prolonged period of time up to 45 weeks in mice. WHV and recombinant WHV-HBV genomes did not trigger effective antibody and T-cell responses to viral proteins. The ability of recombinant WHV constructs to persist in mice is an interesting aspect for the future investigation and may be explored for in vivo gene transfer.     

Paradoxical HBsAg and anti-HBs coexistence among Chronic HBV Infections: Causes and Consequences

Hepatitis B surface antigen (HBsAg) and Hepatitis B surface antibody (anti-HBs) were reported simultaneously among Hepatitis B virus (HBV) infections. HBsAg is a specific indicator of acute or chronic HBV infections, while anti-HBs is a protective antibody reflecting the recovery and immunity of hosts. HBsAg and anti-HBs coexist during seroconversion and then form immune complex, which is rare detected in clinical cases. However, with the promotion of vaccination and the application of various antiviral drugs, along with the rapid development of medical technology, the coexistence of HBsAg and anti-HBs has become more prevalent. Mutations in the viral genomes, immune status and genetic factors of hosts may contribute to the coexistence. Novel HBsAg assays, with higher sensitivity and ability to detect mutations or immune complexes, can also yield HBsAg/anti-HBs coexistence. The discovery of coexistence has shattered the idea of traditional serological patterns and raised questions about the effectiveness of vaccines. Worth noting is that HBsAg/anti-HBs double positivity is strongly associated with progressive liver diseases, especially hepatocellular carcinoma. In conclusion, viral mutations, host factors, and methodology impacts can all lead to the coexistence of HBsAg and anti-HBs. This coexistence is not an indicator of improvement, as an increased risk of adverse clinical outcomes still exists.     

Hepatitis B markers in southern Altaĭ inhabitants of the village of Mendur-Sokkon (the Republic of Altaĭ). HBsAg subtyping in hepatitis B virus isolates with monoclonal antibodies

Blood samples taken from 231 native inhabitants of the village of Mendur-Sokkon located in the Republic of Altai (South-Western Siberia, Russia) were tested for the presence of virus hepatitis B (HBV) markers. 31 samples (13.4%) were found to contain HBsAg, 111 samples (48.05%) were found to contain total anti-HBc antibodies, 123 samples (53.24%) were found to contain anti-HBs antibodies and 15 blood samples (6.49%), anti-HBc antibodies without anti-HBs antibodies and HBsAg. The age-dependent distribution of the occurrence of HBV markers among the aboriginal population of the South Altal remained unchanged (69.9 +/- 7.9%) for the last 50 years. The vertical and horizontal routes of HBV transmissions were noted. The data obtained in this study are indicative of a highly endemic character of HBV of the territory of Mendur-Sokkon. HBsAg-positive blood samples were taken for HBsAg subtyping with the use of a panel of monoclonal antibodies. Two subtypes of HBsAg were detected: ayw1-2 and ayw3varB with the occurrence of 92.6% and 7.4%, i.e. distributed in the ratio 25/2.