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.     

Immunoassay detection of hepatitis B surface antigen mutants.

The increasing use of hepatitis B vaccination has had an overwhelming positive impact on the prevention of hepatitis B viral infection. Mutations in the hepatitis B surface antigen (HBsAg) gene occur as a result of vaccine escape mutants, anti-hepatitis B surface antigen immunotherapy, or in chronic hepatitis B viral infection. These mutants may present a challenge to immunoassay detection. Evaluation of the immunodetection of various HBsAg mutants has been sporadic, as the occurrence of these mutants is not common, and sufficient volume of serum samples is difficult to obtain. To investigate mutant detection, recombinant antigens were constructed to reflect mutations described in the literature occurring throughout the S gene. A limited number of serum samples exhibiting discordant immunoassay reactivity were also used to construct recombinant antigens. The evaluation of 25 HBsAg mutants across nine commercial assays of differing formats is described. Mutations affecting immunoassay performance were characterized as occurring mainly in loop 2 of the "a" determinant of HBsAg. It was determined that reagent epitope recognition was more significant for mutant detection than assay format.     

乙肝表面抗原突变体的表达及其初步应用

目的：构建和表达乙肝表面抗原（HBsAg）突变体用于HBsAg抗原性的深入研究。方法：利用定点突变技术构建HBsAg突变体，然后转化毕赤酵母GS115，菌落PCR、高浓度Zeocin抗性筛选鉴定转化子，表达产物经SDS—PAGE分析和Western blot分析，利用AxSYM HBsAg V2（Abbott）酶免试剂盒检测重组表面抗原的活性。结果：通过序列分析确定突变体构建成功，SDS—PAGE显示突变体能在毕赤酵母中有效表达，在Western blot试验中HBsAg突变体被特异性多克隆抗体识别，相对分子质量（Mr）约为38000，AxSYM试剂盒检测结果表明HBsAg突变体具有一定生物活性。结论：利用毕赤酵母表达系统高效表达出具有一定免疫反应性的HBsAg突变体。对于目前市售试剂盒的质量控制和临床应用有较高的实用价值。     

Linearized hepatitis B surface antigen and hepatitis B core-related antigen in the natural history of chronic hepatitis B

Changes in two novel HBV serological markers, linearized hepatitis B surface antigen (HQ-HBsAg) and hepatitis B core-related antigen (HBcrAg), in the natural history of chronic hepatitis B (CHB) have not been well characterized. Serum HQ-HBsAg and HBcrAg levels of 404 Asian treatment-naïve CHB patients were analysed in a cross-sectional manner. Patients were categorized into five groups: immune tolerant (IT group, n = 52), immune clearance (IC group, n = 105), hepatitis B e antigen (HBeAg)-negative hepatitis (ENH group, n = 97), HBeAg-negative quiescent group (ENQ group, n = 95) and CHB with hepatitis B surface antigen (HBsAg) seroclearance (SC group, n = 55). HQ-HBsAg and HBcrAg were measured and correlated with HBV DNA, HBsAg, HBV genotype and clinical parameters. HQ-HBsAg showed good correlation with HBsAg, especially in the ENQ group (r = 0.874, p <0.001). Correlation of HQ-HBsAg with HBV DNA was less prominent and weakest in the ENH group (r = 0.268, p 0.008). HBcrAg correlated best with HBV DNA in the ENQ group (r = 0.537, p <0.001). In the ENQ group, 42.1% of patients had undetectable HBcrAg; this subgroup of patients, when compared with those with detectable HBcrAg, had significantly lower median HBV DNA (3.17/4.48 log IU/mL, p <0.001) and HBsAg (5.05/5.96 log mIU/mL, p <0.001) levels. Forty per cent of the SC group patients had detectable HQ-HBsAg and/or HBcrAg up to 42 months after HBsAg seroclearance. When comparing anti-HBs positivity and median time after HBsAg seroclearance in the SC group with and without detectable HQ-HBsAg/HBcrAg, there was no significant difference (22.7% and 36.4%, respectively, p 0.284, and 76.5 and 93.2 months, respectively, p 0.245). HQ-HBsAg and HBcrAg showed unique patterns of distribution throughout the five disease phases of CHB, including high detectability rates after HBsAg seroclearance, opening up different possibilities for their applicability.     

乙型肝炎表面抗原不同突变体对细胞免疫的影响

目的　研究乙型肝炎表面抗原(HBsAg)不同突变体对细胞免疫的影响。方法　将野生型和变异型HBsAg基因重组质粒NS2 Swt、NS2 S12 6、NS2 S133、NS2 S14 1、NS2 S14 5分别转染CHO细胞,72h收获细胞上清。采用ELISA法检测各组细胞上清preS2 蛋白的表达量。将这些细胞上清刺激PHA活化的人淋巴细胞,MTS法检测不同变异株抗原对淋巴细胞增殖活性以及淋巴细胞分泌IFN γ、IL 10和IL 2的影响。结果　变异和野毒株(wt)各组细胞上清preS2 蛋白的表达量基本一致。变异和wt重组HBsAg刺激T细胞后,其上清MTS显色后的A4 90 值均高于空白组和pCI neo组,说明HBsAg中的蛋白可以促进T细胞增殖;T12 6S氨基酸变异HBsAg能够刺激IFN γ分泌增加;M133T氨基酸变异刺激IL 10分泌增加。结论　这4种乙型肝炎病毒(HBV)变异株感染人体后,机体对它们的细胞免疫反应可能不会有明显的增强或减弱,但也不能忽视T12 6S和M133T变异抗原改变了某些细胞因子的表达,可能对机体细胞免疫造成的潜在影响。     

Association of concurrent hepatitis B surface antigen and antibody to hepatitis B surface antigen with hepatocellular carcinoma in chronic hepatitis B virus infection

Antibody to hepatitis B surface antigen (HBsAg) (anti‐HBs) can exist in patients with chronic hepatitis B virus (HBV) infection. To date, little is known about the association of concurrent HBsAg and anti‐HBs (concurrent HBsAg/ anti‐HBs) with hepatocellular carcinoma (HCC). The aim of this study was to investigate the clinical relevance of concurrent HBsAg/anti‐HBs with preS deletion mutations and HCC in chronic HBV infection. A total of 755 patients with chronic HBV infection were included consecutively at a tertiary center. Logistic regression analysis was used to identify risk factors for HCC, and serum HBV DNA was amplified, followed by direct sequencing to detect preS deletions. The prevalence of concurrent HBsAg/anti‐HBs was 6.4% (48/755) and all HBVs tested were genotype C. HCC occurred more frequently in the concurrent HBsAg/anti‐HBs group than in the HBsAg only group [22.9% (11/48) vs. 7.9% (56/707), P = 0.002]. In multivariate analyses, age >40 years [odds ratio (OR), 14.712; 95% confidence interval (CI), 4.365–49.579; P < 0.001], male gender (OR 2.431; 95% CI, 1.226–4.820; P = 0.011), decompensated cirrhosis (OR, 3.642; 95% CI, 1.788–7.421; P < 0.001) and concurrent HBsAg/anti‐HBs (OR, 4.336; 95% CI, 1.956–9.613; P < 0.001) were associated independently with HCC. In molecular analysis, preS deletion mutations were more frequent in the concurrent HBsAg/anti‐HBs and HCC groups than in the HBsAg without HCC group (42.3% and 32.5% vs. 11.3%; P = 0.002 and 0.012, respectively). In conclusion, concurrent HBsAg/anti‐HBs is associated with preS deletion mutations and may be one of the risk factors for HCC in chronic HBV infection with genotype C. J. Med. Virol. 81:1531–1538, 2009. © 2009 Wiley‐Liss, Inc.     

Subtyping of hepatitis B surface antigen by radioimmunoassay.

A solid-phase radioimmunoassay used for HBsAg screening of blood donors at the Finnish Red Cross Blood Transfusion Service (FRC-RIA) was modified for subtyping of HBsAg. The anti-HBs coated test tubes of FRC-RIA served as the solid-phase anti-HBs. Monospecific ¹²⁵I-labelled antibodies were prepared by absorption of the labelled anti-HBs used in FRC-RIA with partially purified HBsAg coupled to Sepharose 4B.Labelled anti-y detected 4 ng/ml of ay antigen and 160 ng/ml of ad antigen; the reagent was 40 times more sensitive for the homologous subtype. Labelled anti-d detected 3 ng/ml of ad antigen, but it did not bind to ay antigen. Thus, anti-d was completely monospecific. The standard FRC-RIA was only about 5 times more sensitive than the subtype RIA; 96% of HBsAg positive blood donor samples screened by FRC-RIA could by subtyped by RIA.The important feature of this method is the absorption of the antibodies in the presence of carrier protein after radioactive labelling. This has certain advantages: (1) No extra radioactive labelling is needed for subtyping, because the labelled anti-HBs of standard RIAs for HBsAg can be utilized. (2) Small amounts of labelled monospecific antibodies can be prepared at a time, which promotes economical use of reagents. (3) Only crude antigen preparation (e.g. polyethyleneglycol precipitate from plasma) is needed for the absorption of antibodies.     

Overlapping gene mutations of hepatitis B virus in a chronic hepatitis B patient with hepatitis B surface antigen loss during lamivudine therapy

Disappearance of hepatitis B surface antigens (HBsAg) in chronic hepatitis B usually indicates clearance of hepatitis B virus (HBV) infection. However, false HBsAg negativity with mutations in pre-S2 and 'a' determinant has been reported. It is also known that YMDD mutations decrease the production of HBV and escape detection of serum HBsAg. Here, we report overlapping gene mutations in a patient with HBsAg loss during the lamivudine therapy. After 36 months of lamivudine therapy in a 44-yrold Korean chronic hepatitis B patient, serum HBsAg turned negative while HBV DNA remained positive by a DNA probe method. Nucleotide sequence of serum HBV DNA was compared with the HBV genotype C subtype adr registered in NCBI AF 286594. Deletion of nucleotides 23 to 55 (amino acids 12 to 22) was identified in the pre-S2 region. Sequencing of the 'a' determinant revealed amino acid substitutions as I126S, T131N, M133T, and S136Y. Methionine of rtM204 in the P gene was substituted for isoleucine indicating YIDD mutation (rtM204I). We identified a HBV mutant composed of pre-S2 deletions and 'a' determinant substitutions with YMDD mutation. Our result suggests that false HBsAg negativity can be induced by combination of overlapping gene mutations during the lamivudine therapy.     

Kidney transplantation in hepatitis B surface antigen carriers

Chronic hepatitis B surface antigen (HBsAg) carriers run a high risk of developing chronic liver disease after renal transplantation. To determine the impact of liver disease on long-term morbidity and mortality of HBsAg carriers following kidney transplantation we analyzed 1977 patients, including 76 HBsAg carriers, who underwent renal transplantation during the period 1968–1992. Although the HBsAg carriers had a better 5-year patient and graft survival rate (94% and 83%) than HBsAg-negative patients (87% and 61%), the prognosis was poor after the tenth year of transplantation. Transplant loss is more frequently caused by death of the HBsAg carriers, in contrast to the total population (34% vs 17% for HBsAg-negative patients). Death occurs in 73% of cases due to complications of hepatitis B. In the HBsAg-negative patients, the predominant cause of death is cardiovascular failure (51% vs 11% in HBsAg carriers), whereas only 2% died of liver disease. Kidney transplantation in HBsAg carriers with normal liver function appears to be justified because of rare graft loss due to acute rejection, low early morbidity and mortality, and late onset of fatal hepatic deterioration.Abbreviations HBsAg hepatitis B surface antigen - HBV hepatitis B virus - HBeAg hepatitis B e antigen - GOT glutamic oxaloacetic transaminase - GPT glutamic pyruvate transaminase - GLDH glutamate dehydrogenase - AP alkaline phosphatase - GGT gamma-glutamyl transferase - CHE buturyl cholinesterase - LDH lactate dehydrogenase - HBc hepatitis B core - HDV hepatitis delta virus - HCV hepatitis C virus - CsA cyclosporine A - Aza azathioprine - RIA radioimmunoassay - HD haemodialysis - KTx kidney transplantation - LTx liver transplantation - CMV cytomegalovirus Correspondence to: V Kliem     

Glycopeptide composition of hepatitis B surface antigen

Three major polypeptides of hepatitis B surface antigen (HBsAg), with mol. wt. 22,000 (p22), 27,000 (p27) and 68,000 (p68), were separated by preparative SDS-PAGE. These three peptides as well as intact HBsAg were found to have almost identical amino acid compositions and carbohydrate was detected in p27 and p68 by PAS staining. Papain treatment of p68 produced two distinct peptides p27 and p22. Moreover, when an artificial mixture of p27 and p22 in a ratio of 1:1 was treated with 0.2 M-periodate for 30 min at 37 degrees C, only p22 was detectable. These results suggest that p68 is composed of p27 and p22, and that p27 is a glycosylated product of p22. Thus, from the evidence obtained, it is possible that p22 (22,000 peptide) is the minimum size of the unique hepatitis B virus (HBV) gene product involved.     

The detection of hepatitis B surface antigen by radioimmunoassay.

A comparative study was performed to assess the sensitivity and specificity of counterimmunoelectrophoresis (CIEP) and radioimmunoassay (RIA) for the detection of hepatitis B surface antigen. The 8,823 sera examined included selected reference panels and sera collected from populations with low, moderate and high rates of chronic antigen carriage. Overall, hepatitis B surface antigen was detected in 265 sera by CIEP and in 376 by RIA. As well as detecting 46.4% additional positives, the RIA test detected all CIEP-positive sera; i.e., there were no false negative results. However, 150 sera (1.8% of the total tested) gave a positive result by RIA which was not repeatable on retesting. The explanation for this phenomenon appeared to lie in inadequate washing of the antibody-coated tubes.     

Unusual hepatitis B surface antigen variation in a child immunised against hepatitis B

Perinatal transmission of and infection with hepatitis B (HBV) in early childhood are observed in a small proportion of the offspring of hepatitis B surface antigen (HBsAg)-positive mothers who are vaccinated against HBV immediately after giving birth. The children may be infected by wild-type HBV or by variants with amino acid substitutions in the "a" determinant of HBsAg, particularly at position 145 and, rarely, at positions 120, 126, 129, 131, 141, and 144. Four hundred and forty-six newborn infants of HBsAg-positive mothers in the northeastern part of the Czech Republic received combined active and passive immunisation against HBV. Only one child became an HBsAg carrier. This followed a mild, acute HBV illness in the beginning of the second year of his life. HBV DNA encoding the "a" determinant and surrounding region of HBsAg was sequenced after amplification from the plasma of the child and his mother. The child was infected with variants of HBsAg with substitutions at residues 137 and 139. The virus of the mother had changes at residues 120 and 121. HBV from both child and mother had an unusual substitution at residue 118 and seemed to be of the ayw subdeterminant.     

Fulminant hepatitis in asymptomatic hepatitis B surface antigen carriers in Greece

Eleven male fulminant hepatitis (FH) patients (mean age: 47.7 +/- 16 years) positive for hepatitis B surface antigen (HBsAg) but negative for IgM antibody to hepatitis B core antigen (IgM anti-HBc) were admitted consecutively to the Athens Hospital for Infectious Diseases between May 1981 and November 1983. Because of the absence of IgM anti-HBc, determined by an enzyme immunoassay, these patients were considered to be HBsAg carriers with a superimposed acute hepatitis. Three of the 11 patients received immunosuppressive chemotherapy during the six months before the onset of the acute hepatitis. None of the patients was homosexual or a drug addict. Infection with hepatitis A virus (HAV), hepatitis B virus (HBV), or hepatitis delta virus (HDV) was detected with serologic markers and/or molecular hybridization techniques. Fulminant hepatitis was attributed to spontaneous reactivation of chronic hepatitis B in four patients, chemotherapy-induced reactivation of chronic hepatitis B in three patients, HDV superinfection in one patient and possible superinfection by non-A, non-B agent(s), HDV, or HDV-like agents in three patients. Reactivation of chronic hepatitis B was an important cause of apparent acute hepatitis in heterosexual male HBsAg carriers from an area with a high prevalence of HBV infection.     

Necrosis of the hepatocytes with hepatitis B surface antigen. Occurrence in a chronic hepatitis B surface antigen carrier.

Light and electron microscope finding from a liver of a chronic carrier of hepatitis B surface antigen (HBsAg) showed small lymphocytes and macrophages in close contact with liver cells, partial lysis of variable degrees, lytic necrosis, and the complete loss of a few hepatocytes with HBsAg in the cytoplasm. On the basis of these findings, together with the results from immunofluorescence study, the pathogenesis of hepatitis B is discussed, with emphasis on the importance of host cellular immune response. The cytopathic and cytolytic activities of immunologically activated T lymphocytes against liver cells that have antigenic targets associated with HBsAg at their surface and in the cytoplasm are discussed.     

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.