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Characterization of the reactivity pattern of murine monoclonal antibodies against wild-type hepatitis B surface antigen to G145R and other naturally occurring “A” loop escape mutations
Abstract
The hepatitis B surface antigen (HBsAg) "a" domain harbors major B-cell epitopes. Viruses with mutations in this region emerge after vaccination or during hepatitis B immune globulin (HBIg) prophylaxis. A strain with G145R replacement has been almost invariably isolated as a major escape mutant. We investigated mutant antigen-antibody interactions with direct binding assays. G145R and 16 other naturally occurring recombinant HBsAg mutants were expressed in mammalian Cos-1 cells. The reactivity of a panel of 28 murine anti-hepatitis B surface antigen (anti-HBs) monoclonal antibodies to mutant antigens was measured with enzyme immunoassay and expressed as percentage compared with the wild-type (wt) HBsAg signal for each antibody. All point-mutated proteins displayed diffuse intracellular immunofluorescent labeling corresponding to a secretory pathway. Monoclonal antibodies (mAbs) were classified according to different binding patterns. The effect of mutations on antibody binding differs depending on the amino acid involved and on the location within the "a" loop. As expected, most antibodies had absent or negligible binding (<40%), notably with residue 145 replacements. However, we identified antibodies that reacted with conformational epitopes but nevertheless had adequate reactivity (>40%) with all mutant antigens, including G145R. The effect of G145R was more pronounced than that of G145A. A subgroup of antibodies had substantially increased recognition (>120%) of antigens with mutations in the first loop. We demonstrated that antibodies can be selected or combined that react with all mutants investigated, including G145R. These data offer perspectives for improving anti-HBs-based protection against hepatitis B.
... Those "immune escape" mutants with aa substitutions within a-determinants were found in different clinical settings, including vaccines, transplant patients receiving hyperimmunoglobulins, and immunocompromised patients with HBV reactivation [59][60][61] . Such mutant HBsAg commonly showed reduced binding to anti-HBs antibodies and decreased reactivity in established HBsAg detection assays [59][60][61][62][63][64][65] . ...
... Those "immune escape" mutants with aa substitutions within a-determinants were found in different clinical settings, including vaccines, transplant patients receiving hyperimmunoglobulins, and immunocompromised patients with HBV reactivation [59][60][61] . Such mutant HBsAg commonly showed reduced binding to anti-HBs antibodies and decreased reactivity in established HBsAg detection assays [59][60][61][62][63][64][65] . The most widely known mutation is the sG145R mutation, which has been shown to be replication competent, may persist stably over time, and may be transmitted vertically or horizontally [66][67][68][69] . ...
... These mutations impaired virion secretion and caused lower binding affinity to antibodies used for HBsAg immunoassays [163] . For these reasons, the mutations can render HBsAg undetectable or poorly detected by immunoassays based on monoclonal antibodies against wild-type virus [60,62,65,164] , contributing to some cases of OBI [165][166][167][168][169][170] (Figure 3). ...
Hepatitis B virus (HBV) infection is a global public health concern. HBV causes chronic infection in patients and can lead to liver cirrhosis, hepatocellular carcinoma, and other severe liver diseases. Thus, understanding HBV-related pathogenesis is of particular importance for prevention and clinical intervention. HBV surface antigens are indispensable for HBV virion formation and are useful viral markers for diagnosis and clinical assessment. During chronic HBV infection, HBV genomes may acquire and accumulate mutations and deletions, leading to the expression of defective HBV surface antigens. These defective HBV surface antigens have been found to play important roles in the progression of HBV-associated liver diseases. In this review, we focus our discussion on the nature of defective HBV surface antigen mutations and their contribution to the pathogenesis of fulminant hepatitis B. The relationship between defective surface antigens and occult HBV infection are also discussed.
... The envelope of HBV consists primarily of large (preS1-preS2-S), middle (preS2-S), and small (S) surface proteins, and a host-cellderived lipid membrane. The 226-residue S protein and its glycosylated form (p24/gp27) are the major proteins of the outer envelope of HBV virions [5,7]. The small protein contains the major hydrophilic region (MHR), which spans residues 99-169 and contains several conformational epitopes [22]. ...
... The ''a'' determinant (residues 124-147) is a highly conformational structure composed of two loops held by disulfide bridges (cysteines 124-137 and 138-147 or 149) projecting from the viral surface. Antibodies against this region are thought to confer protection against HBV and are essential for virus clearance [5]. Based on serotyping and nucleotide sequencing of the HBs molecule, HBV has been classified into nine major serotypes and eight genotypes, respectively [4,19]. ...
... Consequently, the ''a'' determinant is the most important target for serological diagnosis, vaccination, and immunoprophylaxis after liver transplantation. High-affinity antibodies against epitopes in the second loop are found in responding vaccinees and are used for hepatitis B immune globulin (HBIG) prophylaxis in liver-graft recipients [5]. ...
The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There is an increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG).
... In the present study, we have characterized eight murine HBsAg-specific mAbs as potential tools for detection of HBsAg mutants. The results presented in this paper and similar papers reported previously [20,30,31] will help to identify the most infl uential mutations relevant to vaccine escape and immunodetection failure. ...
... Reactivity patterns of anti-HBs mAbs with HBsAg mutants HBsAg as highly immunogenic epitopes. The majority of mAbs produced against HBsAg have been shown to recognize conformational epitopes within one of these loops [16,30,31,37] . The tertiary structure created by these disulphide bonds is critical for antigenicity of HBsAg and replacement of cysteine residues at positions 124 and 147 with serine destroys its antigenicity [38] , demonstrating the conformational nature of the epitopes. ...
... Furthermore, recognition of three other mutants, namely PA17D, M5 and BA2.4 having multiple mutations, by these two mAbs makes a distinction between this group of mAbs with other groups. A variety of mutations exist within the first and second loops of "a" determinant of these mutants, including residues 133, 134, 144 and particularly 145, which are critical for binding many of the mAbs [31] . These mutations, however, do not signifi cantly infl uence reactivity of 37C5-S7 and 35C6-S11 mAbs. ...
To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the "a" region.
The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).
Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the "a" determinant at positions 120 (P-->S), 123(T-->N) and 161 (M-->T) were found to affect reactivity of these mAbs.
Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.
... The OD obtained for wt HBsAg reaction was taken as 100%. The signal obtained for each individual mutant at the preset concentration was compared with the wt signal and expressed as percent change of reactivity based on Cooreman et al. 17 The concentration of these mutant forms in the supernatant of transfected CHO cells was measured with commercial ELISA kit (BioELISA HBsAg, Biokit, Barcelona, Spain), which employs polyclonal antibodies for both capture and detection layers and recognizes mutant forms of HBsAg with high specificity and sensitivity. ...
... Therefore, the "a" determinant is a crucial target for diagnosis and immunoprophylaxis. 17 It is assumed that mutation changes immunological specificities of S protein and could lead to propagation of the mutant forms of the virus in the presence of neutralizing antibodies. 17 An amino acid substitution within the "a" determinant, however, neither necessarily impairs the immunogenicity of HBsAg, nor reduces the binding of anti-HBs antibodies. ...
... 17 It is assumed that mutation changes immunological specificities of S protein and could lead to propagation of the mutant forms of the virus in the presence of neutralizing antibodies. 17 An amino acid substitution within the "a" determinant, however, neither necessarily impairs the immunogenicity of HBsAg, nor reduces the binding of anti-HBs antibodies. Zheng et al. have shown that HBsAg G145R is immunogenic and still able to induce mutant-specific antibody response in mice. ...
Antibodies against the "a" determinant of hepatitis B surface antigen (HBsAg) are able to neutralize circulating HBV particles and to prevent HBV infection. It has been proposed, that a single amino acid exchange may allow the virus to escape the immune response. We used a set of monoclonal antibodies (MAbs) to investigate whether a single mutation may account for virus escape from humoral immunity.
Nine murine HBsAg specific MAbs were raised. Reactivity of all antibodies with 14 recombinant mutants of HBsAg was assessed by ELISA. HBV infection of HepaRG cells was used to evaluate viral neutralization capacity of MAbs in vitro.
All MAbs were able to inhibit the establishment of HBV infection in a dose dependent fashion, but recognition of HBsAg variants varied. The MAbs were classified into 3 subgroups based on their pattern of reactivity to the HBsAg variants. Accordingly, three MAbs showed weak reactivity (<40%) to variants with mutations within the first loop of "a" determinant, five MAbs displayed negligible binding to variants with mutations within the second loop and one MAb lost its binding to variants having mutations in both loops of the "a" determinant.
Our results indicate that antibodies against different epitopes of the "a" determinant of HBsAg are able to neutralize HBV. It seems that mutations within a single or a limited number of amino acids within this determinant can hardly result in viral escape. These results have important implications for the development of antibody-based therapies against HBV.
... Most anti-S antibodies target the "a" determinant (residues 124-147) within the immunodominant loop, where mutations are associated with vaccine failure. The most common vaccine escape mutation, G145R, has been shown to impair the binding of antibodies raised against the wild-type (WT) S protein (Chiou et al., 1997;Cooreman et al., 1999;Waters et al., 1992). The rising prevalence of such immune escape mutants since the inception of the universal vaccination programs in Asian countries (Bian et al., 2013;Hsu et al., 2004) raises serious concerns as to whether they will replace the WT virus and thwart the vaccination programs. ...
... Immune escape mutations such as G145R reduce the affinity of the mutant envelope proteins for anti-HBs antibody (Chiou et al., 1997;Cooreman et al., 1999;Waters et al., 1992), thus leading to diagnostic failure, occult HBV infection, vaccine failure and breakthrough infection despite presence of neutralizing antibodies against WT S domain. Since in the present study polyclonal or monoclonal antibodies against WT virus were used for Western blot detection of S protein, ELISA and Abbott Architect i2000 quantification of HBsAg, and immunoprecpitation of virions, caution should be exercised in data interpretation for the immune escape mutants and N-linked glycosylation mutants. ...
Hepatitis B virus (HBV) expresses three co-terminal envelope proteins: large (L), middle (M), and small (S), with the S protein driving the secretion of both virions and subviral particles. Virion secretion requires N-linked glycosylation at N146 in the S domain but can be impaired by immune escape mutations. An M133T mutation creating a novel glycosylation site at N131could rescue virion secretion of N146Q mutant (loss of original glycosylation site) and immune escape mutants such as G145R. Here we demonstrate that other novel N-linked glycosylation sites could rescue virion secretion of the G145R and N146Q mutants to variable extents. Both G145R and N146Q mutations impaired virion secretion through the S protein. The M133T mutation restored virion secretion through the S protein, and could work in trans. Impaired virion secretion was not necessarily associated with a similar block in the secretion of subviral particles.
... Most epidemiological studies in HD patients have been performed using only serological testing of HCV antibodies [16][17][18][19]. In 1993, Bukh and colleagues [20] described the fact that HCV viremia can occur without detection of HCV antibodies, as in patients in advanced phase of CKD due to deficiencies in their immune system. ...
... As shown in different studies [7,16,21,[29][30][31][32][33][34][35][36][37][38][39]and also as resulted in our study, HD patients are at particular high risk for blood borne infections. The prevalence of both HCV and HBV infection is consistently associated with patient age, number of transfused blood products and accuracy of laboratory tests for detection of these infections' markers. ...
Objective
Evaluation of the prevalence of viral hepatitis markers in CKD patients and comparison with the prevalence in family replacement donors as a control group of the general population in Albania.
Methods
64 patients with CKD treated with three or more blood transfusions were evaluated for Hepatitis B and C markers. They were divided in hemodialysed (HD) and non-hemodialysed (Non- HD) patients. We compared the prevalence of HBV and HCV markers founded in these patients with those of blood donors group which includes 1993 subjects divided in two categories: regular blood donors 625 subjects and family replacement donors 1368 subjects, the later being considered as control group.
Result
The prevalence of viral hepatitis B and C markers found in CKD patients were significantly higher than those of the control group (27% and 31% vs 8.5% and 2%). There are no significant differences of HBsAg prevalence in HD and Non-HD patients, but there is a significant one in the presence of anti HCV in HD patients. We also assessed the impact of age and temporal duration of hemodialysis in this prevalence. We confirm that the prevalence of HCV and HBV markers is significantly higher in CKD compared to the general Albanian population.
Conclusions
The amount of blood transfusions and the age of patients are important risk factors for the spread of these infections.
... The expressed HBsAg levels produced by these constructs were evaluated by a polyclonal antibody based commercial detection assay to avoid the influence of these single mutations on their quantification. Appearance of anti-HBs antibodies leads to protection against HBV infection, therefore altered antigenicity of surface mutants could affect most clinical aspects of hepatitis B: chronicity, vaccine efficacy, effectiveness of HBIG immunoprophylaxis posttransplantation and diagnostic accuracy (20). HBV exhibits a mutation rate more than 10-fold higher than other DNA viruses due to lack of proofreading function of reverse transcriptase (21). ...
... Inclusion of the mutant forms of HBsAg within the current standard HBV vaccines might be essential as the new generation vaccines. Even though HBIG is an effective prophylaxis therapy, liver-graft infection with HBV still occurs in about 30% of patients receiving HBIG (20). Additionally, HBIG therapy for long-term can provoke emergence of genetic HBV mutants, which may cause the virus to become resistant to neutralization (32). ...
Background:
The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays.
Objectives:
To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs).
Methods:
Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA.
Results:
Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ''a'' determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs.
Conclusion:
Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.
... A variety of explanations for persistence of HBV DNA in HBsAg-negative individuals have been proposed, including the occurrence of amino acid substitutions in the HBsAg. Several HBsAg variants with reduced reactivity against anti- HBs antibodies have been characterized, most of them with specific mutations in the a determinant of HBsAg [24] [25] [26] [27] [28] ...
... . Viruses with G145R replacement have been almost invariably isolated as a major escape mutant after HBV vaccination [16] [25] [27] [29]. ...
Small hepatitis B virus surface protein (S-HBsAg) variant Y100C has been associated with HBsAg-negative phenotype. To determine whether Y100C substitution yields impaired HBsAg or small amounts of HBsAg that may reduce HBsAg detection by commercial anti-HBsAg antibodies, two eukaryotic expression plasmids, one containing a wild-type S and the other an S gene from a Y100C variant, were constructed and their levels of HBsAg compared by ELISA after transfection of HuH7 cells. Unexpectedly, the extracellular HBsAg levels detected with Y100C plasmid were higher than those observed with the wild-type plasmid, but without statistical significance. We concluded that the Y100C substitution alone did not play a role in reducing HBsAg amounts or HBsAg affinity by commercial ELISA assay. Further studies on
in vitro
replication fitness with the complete genome of HBV isolates displaying or not Y100C substitution may elucidate whether this mutation affects HBV replication and consequently HBsAg production.
... Whereas the aforementioned mechanisms have been proposed for wild-type HBV, the immunological properties of escape mutants have not been explored sufficiently, and most studies have focused on the interaction of mutants with anti-HBsAg antibodies, typically by ELISA [17][18][19][20], and less often using cell lines [21]. However, since pathogenetic changes caused by HBV mutants can differ from those of wild-type HBV, investigation into the effects of HBV mutants on various arms of the immune system is essential for both fundamental and applied science [6]. ...
Immune-escape hepatitis B virus (HBV) mutants play an important role in HBV spread. Recently, the multivalent vaccine Bubo®-Unigep has been developed to protect against both wild-type HBV and the most significant G145R mutant. Here, we compared the effects of recombinant HBsAg antigens, wild-type and mutated at G145R, both included in the new vaccine, on activation of a human high-density culture of peripheral blood mononuclear cells (PBMC) in vitro. The antigens were used either alone or in combination with phytohemagglutinin (PHA). None of the antigens alone affected the expression of CD40, HLA-DR or CD279. Wild-type HBsAg enhanced CD86 and CD69 expression, and induced TNF-α, IL-10, and IFN-γ, regardless of the anti-HBsAg status of donor. In the presence of PHA, wild-type HBsAg had no effect on either of the tested surface markers, but increased IFN-γ and IL-10 and inhibited IL-2. In contrast, the G145R mutant alone did not affect CD86 expression, it induced less CD69, and stimulated IL-2 along with lowering levels of TNF-α, IL-10, and IFN-γ. The G145R mutant also suppressed PHA-induced activation of CD69. The dramatic differences in the immune responses elicited by wild-type HBsAg and the G145R mutant HBsAg suggest distinct adaptive capabilities of the G145R mutant HBV.
... A glycine to arginine substitution (G145R) is the archetypal immune escape mutant that markedly reduces the affinity of anti-HBs binding and is able to survive despite high anti-HBs titers. (65,85) Mutations that affect antigenicity outside of the major "a" determinant region could also affect the binding of circulating antibodies. These "downstream mutations" could affect the conformation of the major HBV S antigenic region and binding of neutralizing antibodies induced by the current vaccine. ...
The hepatitis B virus (HBV) is an important human pathogen. Unvaccinated infants infected through mother-to-child transmission (MTCT) are at >95% risk of developing serum hepatitis B surface antigen-positive chronic hepatitis B (CHB). Despite complete passive-active HBV immunoprophylaxis, approximately 10% of infants born to mothers who are highly viremic develop CHB, and thus maternal treatment with nucleos(t)ide analogs (tenofovir disoproxil fumarate, lamivudine, or telbivudine) is recommended in the third trimester of pregnancy to reduce MTCT risk. Viral rebound usually occurs after stopping treatment and, in the context of maternal immunologic reconstitution postpartum, can also precipitate host immune-mediated hepatic (biochemical) flares. In this article, we review the epidemiology of HBV MTCT, discuss management and potential mechanisms of HBV vertical transmission, and highlight recent studies on virologic and immunologic aspects of hepatitis B in pregnancy and postpartum.
This manuscript reviews epidemiological, clinical, virological and immunological aspects of chronic hepatitis B infection in pregnancy.
... Two main reasons have been proposed for HBV recurrence after LT: the discontinuation of HBIG (10) and the emergence of anti-HBs escape mutants (11,12). The mechanisms by which HBIG protects the transplanted liver against HBV reinfection are still unclear. ...
The use of hepatitis B core antibody (anti-HBc)-positive grafts is one strategy for expanding the donor pool for liver transplantation (LT). The aim of this study was to determine the risk factors associated with hepatitis B virus (HBV) recurrence after living donor LT (LDLT) of anti-HBc-positive grafts. From January 1996 to December 2018, a total of 609 LDLT procedures were performed at our center. A retrospective review was performed for 31 patients (23 males and 8 females; median age = 47 years) who underwent LDLT for HBV-unrelated liver disease from anti-HBc-positive donors. The factors associated with HBV recurrence were evaluated and compared between the HBV recurrence and non-recurrence groups. The median follow-up period after LT was 135 months (range, 6-273 months). Four of 31 patients (12.9%) developed post-LT HBV recurrence. All four cases were HBV-naïve patients (anti-HBc-negative and Hepatitis B surface antibody-negative). The median interval between LDLT and HBV recurrence was 42 months (range, 20-51). The overall actuarial rates of HBV recurrence at 1, 3, 5, 10, and 20 years were 0%, 7.2%, 15.7%, 15.7%, and 15.7%, respectively. Although there were no significant differences between the HBV recurrence and non-recurrence groups, HBV recurrence tended to occur in HBV-naïve recipients (P = 0.093). HBV-naïve status may contribute to HBV recurrence after LDLT for HBV-unrelated liver disease from anti-HBc-positive donors. Careful monitoring for serological HBV markers is needed, particularly in this group.
... The classical G145R immune escape mutant [17,18] was observed in 4.2%, 3.8% and 3.1% of Chinese, Korean and Vietnamese subjects, respectively, and mostly in genotype C-infected subjects. In general, the mutation rate was higher in loop 1, with no significant difference between the genotypes B-and C-infected subjects. ...
Background:
To avoid false negative results, hepatitis B surface antigen (HBsAg) assays need to detect samples with mutations in the immunodominant 'a' determinant region, which vary by ethnographic region.
Objective:
We evaluated the prevalence and type of HBsAg mutations in a hepatitis B virus (HBV)-infected East- and Southeast Asian population, and the diagnostic performance of the Elecsys® HBsAg II Qualitative assay.
Study design:
We analyzed 898 samples from patients with HBV infection from four sites (China [Beijing and Guangzhou], Korea and Vietnam). HBsAg mutations were detected and sequenced using highly sensitive ultra-deep sequencing and compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the 'a' determinant region using the Elecsys® HBsAg II Qualitative assay.
Results:
Overall, 237 distinct amino acid mutations in the major hydrophilic region were identified; mutations were present in 660 of 898 HBV-infected patient samples (73.5%). Within the pool of 237 distinct mutations, the majority of the amino acid mutations were found in HBV genotype C (64.8%). We identified 25 previously unknown distinct mutations, mostly prevalent in genotype C-infected Korean patients (n = 18) followed by Chinese (n = 12) patients. All 898 samples were correctly identified by the Elecsys® HBsAg II Qualitative assay.
Conclusions:
We observed 237 distinct (including 25 novel) mutations, demonstrating the complexity of HBsAg variants in HBV-infected East- and Southeast Asian patients. The Elecsys® HBsAg II Qualitative assay can reliably detect HBV-positive samples and is suitable for routine diagnostic use in East and Southeast Asia.
... Reports have shown that during HBIG therapy, mutation at residue 144 and 145 of HBsAg were emerged and predominated. Apparently HBIG puts selection pressure on the ''a'' determinant (Cooreman et al., 1999;Protzer-Knolle et al., 1998). HBsAg escape mutants can also occur in acute hepatitis B patients and are quite frequently found in individuals with occult hepatitis B (Allain et al., 2013;Aragri et al., 2016;Gerlich et al., 2010). ...
Hepatitis B virus (HBV) infection is a global burden on the health-care system and is considered as the tenth leading cause of death in the world. Over 248 million patients are currently suffering from chronic HBV infection worldwide and annual mortality rate of this infection is 686000. The “a” determinant is a hydrophilic region present in all antigenic subtypes of hepatitis B surface antigen (HBsAg), and antibodies against this region can neutralize the virus and are protective against all subtypes. We have recently generated a murine anti-HBs monoclonal antibody (4G4), which can neutralize HBV infection in HepaRG cells and recognize most of the escape mutant forms of HBsAg. Here, we describe the production and characterization of the chimeric human-murine antibody 4G4 (c-4G4). Variable region genes of heavy and light chains of the m-4G4 were cloned and fused to constant regions of human kappa and IgG1 by splice overlap extension (SOE) PCR. The chimeric antibody was expressed in Chinese Hamster Ovary (CHO)-K1 cells and purified from culture supernatant. Competition ELISA proved that both antibodies bind the same epitope within HBsAg. Antigen-binding studies using ELISA and Western blot showed that c-4G4 has retained the affinity and specificity of the parental murine antibody, and displayed a similar pattern of reactivity to 13 escape mutant forms of HBsAg. Both, the parental and c-4G4 showed a comparably high HBV neutralization capacity in cell culture even at the lowest concentration (0.6μg/ml). Due to the ability of c-4G4 to recognize most of the sub-genotypes and escape mutants of HBsAg, this antibody either alone or in combination with other anti-HBs antibodies could be considered as a potent alternative for Hepatitis B immune globulin (HBIG) as an HBV infection prophylactic or for passive immunotherapy against HBV infection.
... 36,37 According to previous studies, it is possible that the antibodies in HBIG predominantly recognize the conformational "a" determinant and/or second loop epitope. 36,40,45 Most mAbs against the abovementioned HBV surfaceexposed epitopes could neutralize HBV infection in vitro to various degrees. 41 However, their therapeutic uses still need to evaluated in vivo. ...
The currently available drugs to treat hepatitis B virus (HBV) infection include interferons and nucleos(t)ide analogues, which can only induce disease remission and are inefficient for the functional cure of patients with chronic HBV infection (CHB). Since high titers of circulating hepatitis B surface antigen (HBsAg) may be essential to exhaust the host anti-HBV immune response and they cannot be significantly reduced by current drugs, new antiviral strategies aiming to suppress serum hepatitis B surface antigen (HBsAg) could help restore virus-specific immune responses and promote the eradication of the virus. As an alternative strategy, immunotherapy with HBsAg-specific antibodies has shown some direct HBsAg suppression effects in several preclinical and clinical trial studies. However, most previously described HBsAg-specific antibodies only had very short-term HBsAg suppression effects in CHB patients and animal models mimicking persistent HBV infection. More-potent antibodies with long-lasting HBsAg clearance effects are required for the development of the clinical application of antibody-mediated immunotherapy for CHB treatment. Our recent study described a novel mAb E6F6 that targets a unique epitope on HBsAg. It could durably suppress the levels of HBsAg and HBV DNA via Fcγ receptor-dependent phagocytosis in vivo. In this commentary, we summarize the current research progress, including the therapeutic roles and mechanisms of antibody-mediated HBV clearance as well as the epitope-determined therapeutic potency of the antibody. These insights may provide some clues and guidance to facilitate the development of therapeutic antibodies against persistent viral infection.
... This finding was supported by other results from molecular dynamic and docking studies. The lower binding affinity between G145R and MAb12 compared with the wild-type protein indicated the intrinsic potential of this mutant to escape HBsAg neutralization using specific antibodies, as reported in previous studies (52)(53)(54). According to the detailed figures of amino acid interactions revealed by DIMPLOT, the substitution of Gly with Arg leads to a reduction in the total number of H-bonds between the HBsAg and its monoclonal antibody. ...
Background:
Vaccine-escaped hepatitis B virus (HBV) mutations occur within the "a" determinant area, which is located in the major hydrophilic region (MHR) of the hepatitis B surface antigen (HBsAg) protein. It is now well established that the common G145R mutation is highly capable of escaping from HBsAg immune recognition. However, the impacts of this mutation on the structure and immunogenic activity of HBsAg have been poorly investigated.
Objectives:
The present study analyzed the effects of the G145R mutation on the structure and immunogenic activity of the HBsAg.
Materials and methods:
Three-dimensional (3D) structure of HBsAg for both the wild-type and G145R mutant were predicted and refined using several web tools. After quantitative evaluations, the effects of the G145R mutation on the secondary and 3D structures of the HBsAg were investigated. In parallel, the immunogenic activity of the wild-type and mutant HBsAg was also analyzed using a ClusPro docking server as well as the IEDB web tool. Further analyses were performed via molecular dynamics (MD) simulations using the GROMACS v5.0.2 simulation package.
Results:
The G145R mutation causes a considerable reduction in the immunogenic activity of the HBsAg through a conformational change in the HBsAg antigenic loops. This mutation inserts a new β-strand in the "a" determinant region of the HBsAg, leading to a reduced binding affinity to its monoclonal antibody, MAb12. The G145R mutation also increased the compactness and stability of the HBsAg by enhancing the rigidity of the "a" determinant.
Conclusions:
These data will be beneficial for designing more advanced antibodies for the recognition of the HBsAg in diagnostics. In addition, the results of this study may assist in the design or development of more effective hepatitis B vaccines.
... However, single amino acid substitutions such as G145R could markedly reduce the affinity of anti-S antibodies [139][140], and such immune escape mutants could survive the rising anti-HBs antibody better than wild-type virus. Such mutants could contribute to occult HBV infection, which is defined by presence of HBV DNA but no detectable HBsAg [141]. ...
Chronic infection with hepatitis B virus (HBV) greatly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). HBV isolates worldwide can be divided into ten genotypes. Moreover, the immune clearance phase selects for mutations in different parts of the viral genome. The outcome of HBV infection is shaped by the complex interplay of the mode of transmission, host genetic factors, viral genotype and adaptive mutations, as well as environmental factors. Core promoter mutations and mutations abolishing hepatitis B e antigen (HBeAg) expression have been implicated in acute liver failure, while genotypes B, C, subgenotype A1, core promoter mutations, preS deletions, C-terminal truncation of envelope proteins, and spliced pregenomic RNA are associated with HCC development. Our efforts to treat and prevent HBV infection are hampered by the emergence of drug resistant mutants and vaccine escape mutants. This paper provides an overview of the HBV life cycle, followed by review of HBV genotypes and mutants in terms of their biological properties and clinical significance.
... A number of HBsAg variants (vtHBsAg) with aa substitutions within or around the "a" deter-minant have been identified in patients. Some of these may have a reduced affinity for anti-HBs antibodies, thus facilitating escape from neutralization, subsequently leading to diagnostic failure or breakthrough of the HBV infection (Chiou et al., 1997;Cooreman et al., 1999;Carman, 1997). Moreover, such aa substitutions in vtHBsAg can affect HBsAg assembly and secretion, virion formation, and HBV infectivity (Wu et al., 2010;Ito et al., 2010;Wu et al., 2012). ...
... Among the MHR mutations related to vaccine escape, the mutation of glycine to arginine in the 145th codon of HBsAg (i.e., G145R) is detected most frequently worldwide [70,71]. However, the G145R mutation was not observed in any of the 41 Korean patients with occult infections in our previous report. ...
Occult hepatitis B virus infection (HBV) is characterized by HBV DNA positivity but HBV surface antigen (HBsAg) negativity. Occult HBV infection is associated with a risk of HBV transmission through blood transfusion, hemodialysis, and liver transplantation. Furthermore, occult HBV infection contributes to the development of cirrhosis and hepatocellular carcinoma. We recently reported the characteristic molecular features of mutations in the preS/S regions among Korean individuals with occult infections caused by HBV genotype C2; the variants of preS and S related to severe liver diseases among chronically infected patients were also responsible for the majority of HBV occult infections. We also reported that HBsAg variants from occult-infected Korean individuals exhibit lower HBsAg secretion capacity but not reduced HBV DNA levels. In addition, these variants exhibit increased ROS-inducing capacity compared with the wild-type strain, linking HBV occult infections to liver cell damage. Taken together, our previous reports suggest the transmission potential of distinct HBV occult infection-related variants in South Korea.
... The transfection of the uncut full-length PCR clones could permit the very efficient expression of viral envelope proteins. As mentioned previously, this increased sensitivity was useful to study immune escape mutants, which harbor amino acid substitutions in the S protein that abolish or diminish its detection by antibodies raised against wild-type protein [31][32][33]. In addition, occult HBV infection also has been attributed to infection by such immune escape mutants [34,35]. ...
Studies on molecular mechanisms of the persist infection of hepatitis B virus have been hampered by a lack of a robust animal model. We successfully established a simple, versatile, and reproducible HBV persist infection model in vitro and in vivo with the circularized HBV DNA. The cells and mice were transfected or injected with circularized HBV DNA and pAAV/HBV1.2, respectively. At the indicated time, the cells, supernatants, serum samples, and liver tissues were collected for virological and serological detection. Both in vitro and in vivo, the circularized HBV DNA and pAAV/HBV1.2 could replicate and transcribe efficiently, but the infection effect of the former was superior to the latter (p < 0.05). The injection of circularized HBV genome DNA into the mice robustly supported HBV infection and approximately 80% of HBV infected mice established persistent infection for at least 10 weeks. This study demonstrated that the infection efficiency and replication ability of the circularized structure of HBV DNA overmatched that of the expression plasmid containing the linear structure of HBV DNA in vitro and in vivo. Meanwhile, this research results could provide useful tools and methodology for further study of pathogenic mechanisms and potential antiviral treatments of human chronic HBV infection in vitro and in vivo.
... Interestingly, Khattab et al. found, mutations in the "a" determinant of the S gene, including the G145R mutation, in all their 4 co-infected patients, which may have explained the lack of detection of serum HBsAg by conventional serology assays [12]. This is consistent with data showing that these mutations may affect the binding of anti-HBs monoclonal antibodies to the "a" determinant of the surface protein [19]. Moreover, quantification of HBV DNA by a real-time PCR assay also showed relatively low levels of circulating virus, which may have contributed to the lack of detection of HBsAg in serum. ...
ABSTRACT Chronic hepatitis C virus (HCV) infection combined with occult hepatitis B virus (HBV) infection
has been associated with increased risk of hepatitis, cirrhosis and hepatocellular carcinoma. This study aimed
to determine the prevalence of occult HBV infection among Egyptian chronic HCV patients, the genotype and
occurrence of surface gene mutations of HBV and the impact of co-infection on early response to treatment. The
study enrolled 162 chronic HCV patients from Ismailia Fever Hospital, Egypt, who were HBV surface antigennegative.
All patients were given clinical assessment and biochemical, histological and virological examinations.
HBV-DNA was detectable in sera from 3 patients out of the 40 patients who were positive for hepatitis B core
antibody. These 3 patients were responsive to combination therapy at treatment week 12; only 1 of them had
discontinued therapy by week 24. HBV genotype D was the only detectable genotype in those patients, with
absence of “a” determinant mutations among those isolates.
... The mutations diminish the binding of antibodies raised against wild-type S protein to virions and subviral particles. [148][149][150] The former causes breakthrough infections, while the latter leads to diagnostic failure. Presently, the threat posed by immune escape mutants remains uncertain. ...
Hepatitis B virus (HBV) causes a chronic infection in 350 million people worldwide and greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. The majority of chronic HBV carriers live in Asia. HBV can be divided into eight genotypes with unique geographic distributions. Mutations accumulate during chronic infection or in response to external pressure. Because HBV is an RNA-DNA virus the emergence of drug resistance and vaccine escape mutants has become an important clinical and public health concern. Here, we provide an overview of the molecular biology of the HBV life cycle and an evaluation of the changing role of hepatitis B e antigen (HBeAg) at different stages of infection. The impact of viral genotypes and mutations/deletions in the precore, core promoter, preS, and S gene on the establishment of chronic infection, development of fulminant hepatitis and liver cancer is discussed. Because HBV is prone to mutations, the biological properties of drug-resistant and vaccine escape mutants are also explored.
... The mutations diminish the binding of antibodies raised against wild-type S protein to virions and subviral particles. [148][149][150] The former causes breakthrough infections, while the latter leads to diagnostic failure. Presently, the threat posed by immune escape mutants remains uncertain. ...
Hepatitis B virus (HBV) causes a chronic infection in 350 million people worldwide and greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. The majority of chronic HBV carriers live in Asia. HBV can be divided into eight genotypes with unique geographic distributions. Mutations accumulate during chronic infection or in response to external pressure. Because HBV is an RNA-DNA virus the emergence of drug resistance and vaccine escape mutants has become an important clinical and public health concern. Here, we provide an overview of the molecular biology of the HBV life cycle and an evaluation of the changing role of hepatitis B e antigen (HBeAg) at different stages of infection. The impact of viral genotypes and mutations/deletions in the precore, core promoter, preS, and S gene on the establishment of chronic infection, development of fulminant hepatitis and liver cancer is discussed. Because HBV is prone to mutations, the biological properties of drug-resistant and vaccine escape mutants are also explored.
... WHO aims at controlling hepatitis B virus (HBV) worldwide, to decrease the incidence of HBV-related chronic liver disease, cirrhosis, and hepatocellular carcinoma, by integrating hepatitis B vaccination into routine infant (and possibly adolescent) immunization programs. Hepatitis B vaccination is safe and effective, although breakthrough infections occasionally occur in vaccinees [1][2][3]. The appearance of anti-HBs neutralizing antibodies is usually associated with patient recovery. ...
Background & Aims: Occult hepatitis B virus (HBV) infection is a well-recognized clinical entity characterized by the detection of HBV DNA in serum and/or liver in the absence of detectable hepatitis B surface antigen (HBsAg). The frequency of the diagnosis depends on the relative sensitivity of both HBsAg and HBV DNA assays. We aimed at determining the prevalence of occult HBV infection in a high risk group of children who developed HBV infection despite immunoprophylaxis. Methods: The sera of 75 children born to HBsAg-positive mothers previously immunized by HBIG and prophylaxic vaccine regimen were assayed for HBV DNA by real-time PCR. Subsequently, the samples were tested using a sensitive standard PCR, with an independent set of primers for all HBV genes, and analyzed by direct sequencing. Results: HBV DNA was detected in 21/75 (28%) children, and ranged between 77 and 9240 copies/ml. All were positive for anti-HBs. Five (24%) children were found to be positive for anti-HBc, while anti-HBc-only positive individuals were not observed. Eight isolates (38%) did not carry any mutation. Thirteen infected children (62%) had at least one mutation in regions known to be involved in functional and/or immune epitope activity. Ten had G145R mutations. Conclusions: HBV occult infection seems to be relatively frequent in immunized children born to HBsAg-positive mothers. HBsAg negativity is not sufficient to completely exclude HBV DNA presence. These findings emphasize the importance of considering occult HBV infection in hypo-endemic areas.
... An important serological marker of HBV infection is hepatitis B surface antigen (HBsAg), the collective term for viral envelope proteins present on virions and the large excess of subviral particles. The immune escape mutations can render HBsAg undetectable or poorly detected by immunoassays based on monoclonal antibodies against wild-type virus (15)(16)(17)(18), contributing to some cases of "occult HBV infection" (lack of detectable HBsAg despite positive HBV DNA) (8,(19)(20)(21)(22)(23). ...
Hepatitis B virus immune escape mutants have been associated with vaccine failure and reinfection of grafted liver despite
immune prophylaxis, but their biological properties remain largely unknown. Transfection of 20 such mutants in a human hepatoma
cell line identified many with severe impairment in virion secretion, which can be rescued to various extents by coexpression
of wild-type envelope proteins or introduction of a novel glycosylation site. Consistent with their role in maintaining intra-
or intermolecular disulfide bonds, cysteine residues within the “a” determinant are critical for virion secretion.
... Further analysis suggested that the binding ability of this mAb with IEM rHBsAg had some of the following characteristics: (1) This mAb could detect more (12/15) IEM rHBsAgs than the mAbs previously reported and its reaction degree showed an evident character that was either high or low. In addition, its capability of detecting IEM rHBsAg was better than that of commercial kits [23,24]. ...
Using a standard cellular fusion technique and indirect enzyme-linked immunosorbent assay (ELISA), a hybridoma cell line strain secreting anti-HBs monoclonal antibody (mAb) (defined G6 mAb) was obtained. The cells grew and secreted mAb stably. Antibody titers in the culture supernatant and ascites were 2.048×106 and 4.096×106, respectively. By applying the anti-HBs G6 mAb and horseradish peroxidase (HRP)-labeled goat anti-HBs antibody, we developed a sandwich ELISA (defined G6m ELISA) for detecting both wild-type and immune escape mutant HBsAgs (IEM HBsAg). The assay was performed to detect 17 species of genome recombinant expression HBsAg, including two wild-type species and 15 IEM HBsAg species, which varied in the “a” determinant, in a group of patients infected with hepatitis B virus (HBV). The patients previously had a lower ELISA detection signal [(absorbance of patients/absorbance of normal people (P/N): 1.0–4.5)]. The results demonstrated that the sensitivity of this assay to wild-type HBsAg was no less than 0.125 μg/L; 12 of 15 IEM HBsAg species (P/N⩾2.5) were positive for G6 mAb. Of the positive IEM HBsAg species, two had a low absorbance value at 450 nm (A
450), one had an intermediate A450 value and nine had a high A
450 value, which was 7.55%(mean), 59.4%and 92.1%–109.4% of the wild-type A
450 value, respectively. The two species with low A
450 value and the three negative species mutated at the bases 120–124 in the first loop of the HBV “a” determinant. Using the G6 ELISA and two commercial ELISA kits (A and B), 177 patients were tested. The G6 ELISA had a significantly higher detection rate than either commercial ELISAs (19.21% vs 14.89% and 6.21%, respectively; P<0.01, P<0.05, respectively).
... WHO aims at controlling hepatitis B virus (HBV) worldwide, to decrease the incidence of HBV-related chronic liver disease, cirrhosis, and hepatocellular carcinoma, by integrating hepatitis B vaccination into routine infant (and possibly adolescent) immunization programs. Hepatitis B vaccination is safe and effective, although breakthrough infections occasionally occur in vaccinees [1][2][3]. The appearance of anti-HBs neutralizing antibodies is usually associated with patient recovery. ...
Occult hepatitis B virus (HBV) infection is a well-recognized clinical entity characterized by the detection of HBV DNA in serum and/or liver in the absence of detectable hepatitis B surface antigen (HBsAg). The frequency of the diagnosis depends on the relative sensitivity of both HBsAg and HBV DNA assays. We aimed at determining the prevalence of occult HBV infection in a high risk group of children who developed HBV infection despite immunoprophylaxis.
The sera of 75 children born to HBsAg-positive mothers previously immunized by HBIG and prophylaxic vaccine regimen were assayed for HBV DNA by real-time PCR. Subsequently, the samples were tested using a sensitive standard PCR, with an independent set of primers for all HBV genes, and analyzed by direct sequencing.
HBV DNA was detected in 21/75 (28%) children, and ranged between 77 and 9240 copies/ml. All were positive for anti-HBs. Five (24%) children were found to be positive for anti-HBc, while anti-HBc-only positive individuals were not observed. Eight isolates (38%) did not carry any mutation. Thirteen infected children (62%) had at least one mutation in regions known to be involved in functional and/or immune epitope activity. Ten had G145R mutations.
HBV occult infection seems to be relatively frequent in immunized children born to HBsAg-positive mothers. HBsAg negativity is not sufficient to completely exclude HBV DNA presence. These findings emphasize the importance of considering occult HBV infection in hypo-endemic areas.
... Therefore, such variant HBsAgs (vtHBsAgs) are of great importance for the diagnosis of HBV infection and vaccine development. vtHBsAgs may have a reduced ability to bind anti-HBs antibodies, thus escaping neutralization (3,6). Such amino acid substitutions in vtHBsAg could affect HBsAg assembly and secretion, virion formation, and HBV infectivity (14). ...
A variety of amino acid substitutions, such as K122I and G145R, have been identified around or within the a determinant of hepatitis B surface antigen (HBsAg), impair HBsAg secretion and antibody binding, and may be responsible for immune escape in patients. In this study, we examined how different substitutions at amino acid positions 122 and 145 of HBsAg influence HBsAg expression, secretion, and recognition by anti-HBs antibodies. The results showed that the hydrophobicity, the presence of the phenyl group, and the charges in the side chain of the amino acid residues at position 145 reduced HBsAg secretion and impaired reactivity with anti-HBs antibodies. Only the substitution K122I at position 122 affected HBsAg secretion and recognition by anti-HBs antibodies. Genetic immunization in mice demonstrated that the priming of anti-HBs antibody response was strongly impaired by the substitutions K122I, G145R, and others, like G145I, G145W, and G145E. Mice preimmunized with wild-type HBsAg (wtHBsAg) or variant HBsAg (vtHBsAg) were challenged by hydrodynamic injection (HI) with a replication-competent hepatitis B virus (HBV) clone. HBsAg persisted in peripheral blood for at least 3 days after HI in mice preimmunized with vtHBsAg but was undetectable in mice preimmunized with wtHBsAg, indicating that vtHBsAgs fail to induce proper immune responses for efficient HBsAg clearance. In conclusion, the biochemical properties of amino acid residues at positions 122 and 145 of HBsAg have a major effect on antigenicity and immunogenicity. In addition, the presence of proper anti-HBs antibodies is indispensable for the neutralization and clearance of HBsAg during HBV infection.
... Of various mutants with the a determinant, the mutant with a single mutation at the 145th amino acid of the hepatitis B surface antigen (HBsAg) has been frequently reported to cause the failure of prophylaxis in mother-to-child transmission [5][6][7][8][9][10]. Thus, it is indispensable to clarify the frequency of the a determinant mutants when developing the future vaccine strategy in Japan. ...
Hepatitis B virus (HBV) can have mutations that include the a determinant, which causes breakthrough infection. In particular, a single mutation at amino acid 145 of the surface protein (G145) is frequently reported in the failure of prophylactic treatment. The aim of this study was to evaluate the frequency of the a determinant mutants, especially the G145 variant, in Japan, where universal vaccination has not been adopted.
The present study was a retrospective study. The study cohorts were defined as follows: group 1, children with failure to prevent mother-to-child transmission despite immunoprophylaxis (n = 18, male/female = 8/10, age 1-14 years; median 6 years); group 2, HBV carriers who had not received vaccination or hepatitis B immunoglobulin (n = 107, male/female = 107, age 1-52 years; median 16 years). To detect the G145R and G145A mutants in patients, we designed 3 probes for real-time PCR. We also performed direct sequencing and cloning of PCR products.
By mutant-specific real-time PCR, one subject (5.6%) was positive for the G145R mutant in group 1, while the G145 mutant was undetectable in group 2. The a determinant mutants were detected in one (5.6%) of the group 1 subjects and 10 (9.3%) of the group 2 subjects using direct sequencing, but direct sequencing did not reveal the G145 mutant as a predominant strain in the two groups. However, the subject who was positive according to the mutant-specific real-time PCR in group 1 had overlapped peaks at nt 587 in the electropherogram. In group 2, 11 patients had overlapped peaks at nt 587 in the electropherogram. Cloning of PCR products allowed detection of the G145R mutant as a minor strain in 7 (group 1: 1 subject, group 2: 6 subjects) of 12 subjects who had overlapped peaks at nt 587 in the electropherogram.
The frequency of the a determinant mutants was not high in Japan. However, the G145R mutant was often present as a minor population in children and adults. HBV carriers might have the a determinant mutants as a minor form.
... In our study, both assays adequately detected serum samples among patients harboring mutations on the YMDD motif. Other amino acid changes at positions s120 and s145 are also known to change antigenicity in the "a" determinant of the HBsAg (5,21), with previous evidence suggesting higher levels of between-assay disagreement with mutations at position s120 (31). Interestingly, these mutations were the only factor overall for which the Elecsys assay quantified HBsAg at lower levels than the Architect. ...
Hepatitis B surface antigen (HBsAg) quantification has been steadily gaining interest as a clinical marker of therapeutic efficacy, for which two commercial assays are currently available: Architect HBsAg QT (Architect) and Elecsys HBsAg II (Elecsys). HBsAg quantification was evaluated using both assays in 126 human immunodeficiency virus (HIV) and hepatitis B virus (HBV)-coinfected patients initiating treatment with tenofovir dipivoxil fumarate. Linear regression and correlation were used to establish the relationship between the two methods. Bland-Altman analysis was performed to determine mean between-assay difference and limits of agreement (LOA) (±2 standard deviations [SD]) both overall and stratified on HBV (hepatitis B envelope antigen [HBeAg] status, replication, genotype, HBV mutants) or HIV (CD4(+) cell count) cofactors. There was a significant correlation between Elecsys and Architect assays (correlation coefficient, r = 0.959; P < 0.001). HBsAg quantification using the Elecsys assay was on average 0.200 log(10) IU/ml (LOA, -0.500, 0.800) higher than that using Architect, which was consistent across levels of CD4(+) cell count, presence of precore and YMDD mutations, and HBeAg status. A slightly larger mean between-assay difference was observed with genotypes A and G (0.196 and 0.201, respectively) versus HBV genotypes D and E (0.036 and 0.030, respectively). Mutations on the S region at position s120/s145 were the only determinant in which the mean between-assay difference in HBsAg quantification was lower than the null value (-0.078). In conclusion, the Elecsys assay, with automatic on-board dilution, is capable of quantifying serum HBsAg levels in HIV-HBV-coinfected patients, with very high correlation with the Architect assay.
... At any rate, the transfection of the uncut full-length PCR clones will permit the very efficient expression of viral envelope proteins. This increased sensitivity is useful to study immune escape mutants, which harbor amino acid substitutions in the S protein that diminish or abolish its detection by antibodies raised against wild-type protein (4,6,33). In addition, occult HBV infection also has been attributed to infection by such immune escape mutants (8,16). ...
Different hepatitis B virus (HBV) genotypes and variants are associated with different clinical outcomes and/or response to
antiviral therapy, yet the comparison of the in vitro replication capacity of a large number of clinical isolates remains technically challenging and time-consuming. Although
the full-length HBV genome can be amplified from high-titer blood samples by PCR using High Fidelityplus DNA polymerase and primers targeting the conserved precore region, the HBV clones thus generated are replication deficient
due to the inability to generate the terminally redundant pregenomic RNA essential for genome replication. The transfection
experiment is further complicated by PCR errors and the presence of viral quasispecies. A previous study found that the precise
removal of non-HBV sequence by SapI digestion led to HBV replication in transfected cells, possibly due to low-level genome
circularization by a cellular enzyme. We released HBV genome from the cloning vector using BspQI, an inexpensive isoschizomer
of SapI, and increased the efficiency of genome replication by an extra step of in vitro DNA ligation. The uncut plasmid DNA can be used for transfection if the sole purpose is to study envelope protein expression.
We found significant PCR errors associated with the High Fidelityplus DNA polymerase, which could be greatly diminished using Phusion DNA polymerase or masked by the use of a clone pool. The reduced PCR error and modified enzymatic steps prior to transfection
should facilitate a more widespread functional characterization of clinical HBV isolates, while the clone pool approach is
useful for samples with significant sequence heterogeneity.
Antibodies against hepatitis B virus S protein can protect against hepatitis B virus (HBV) infection. Therefore, hepatitis B immunoglobulin (HBIG), which contains HBsAb, is used clinically as a therapy for HBV infection. In this study, we obtained a series of monoclonal antibodies that recognize multiple HBV genotypes. All the antibodies recognized conformational epitopes of S protein, but not linear epitopes. Several antibodies neutralized HBV infection and exhibited strong affinities and neutralizing activities. Antigenic epitope analysis demonstrated that they recognized residue Ile152 of S protein, which is localized outside the "a" determinant. Ile152 is highly conserved, and a mutation in this residue resulted in reduced expression of large hepatitis B surface proteins (L protein), suggesting that the amino acid at this position is involved in the expression of L protein. In addition, the antibodies neutralized the infection of hepatitis D virus possessing a Gly145 mutation to Arg in S protein, which is a well-known escape mutation against HBIG treatment. Using mouse monoclonal antibodies, we successfully established a humanized antibody possessing affinities and neutralizing activities similar to those of the original mouse antibody. The antibodies generated in this study may have potential for use in alternative antibody therapies for HBV infection. This article is protected by copyright. All rights reserved.
We investigated the incidence of acute hepatitis B (AH-B) infection in the metropolitan area from 1994 to 2017. A total of 246 AH-B cases were recorded. The number of patients with AH-B increased between 1994 and 2004 but decreased after 2010. After initiating the observation period, the incidence of infections with genotype (GT) A gradually increased and accounted for most cases after 2006. Infections were also observed in young men; moreover, sexual transmission was frequently observed. Among 166 patients who were continuously observed, 5 developed chronic infections (GTA, 3 patients; GTC, 1 patient; and GTH, 1 patient). The evaluation of amino acid polymorphisms in the a-determinant region revealed that seven patients had amino acid substitutions. Continuous monitoring of the incidence and characterization of AH-B is required, and education regarding the importance of HB vaccination is necessary for controlling this viral infection.
Objective:
To investigate the relationship between the failure of prevention of hepatitis B virus Mother-to-Child transmission and HBV serological pattern, viral load as well as HBV genotypes.
Methods:
2765 pairs of mother-infant matched samples were collected. These pregnant women were HBsAg positive and delivered at hospital from January 1st, 2011 to June 30th, 2011. Of these samples, 26 pairs of sera samples were randomly selected from 114 pairs of samples which failed in the prevention of hepatitis B virus Mother-to-Child transmission. Serological tests, viral load and genotype detection were performed for further analysis. Additionally, the selected subjects were followed and tested again in 2014.
Results:
HBeAg positive rates were 76.92% and 69.23% in mother group and infant group respectively, showed no statistical difference. The average HBV DNA levels were >2×10(5)IU/ml in both mother group and infant group. Genotype analysis revealed that 11 pairs of mother-infant matched samples belonged to C gene type and another 11 pairs were B gene type. Different genotypes were observed in 4 pairs of mother-infant matched samples.
Conclusion:
HbeAg positive and high HBV DNA level were two major risk factors of HBV mother to child transmission. Additionally, nosocomial infection was another potential way of HBV vertical transmission, especially in remote area of Yunnan province.
The division of hepatitis B virus (HBV) isolated from different geographical regions into genotypes and subtypes has long been recognized. This division is based on amino acid variation in the hepatitis B surface antigen (HBsAg)-encoding region, and more specifically the hydrophilic region that constitutes the a antigenic determinant. This same region constitutes the major neutralizing epitope recognized by natural infection- and vaccine-induced antibodies. However, a number of mutations have been described in different clinical settings that allow the virus to evade the neutralizing immune response. This chapter aims to review the current level of knowledge regarding HBsAg variants.
Background:
Hepatitis B virus (HBV) surface antigen (HBsAg) induces a vigorous neutralizing antibody response, which causes effective protection against HBV infection. Little is known about the profile of variable region genes of immunoglobuline heavy (VH) and light (VL) chains rearranged in anti-HBs antibodies, and also the possible association of this profile with specificity pattern of these antibodies to mutant forms of HBsAg.
Aims:
The present study determined the nucleotide sequence of VH and VL genes of mouse monoclonal antibodies (MAbs) generated against HBsAg.
Methods:
Hybridoma clones secreting anti-HBsAg MAbs were developed from hyperimmunized Balb/c mice. VH and VL gene sequences of all MAbs were determined by amplifying the genes using a panel of VH and VL family specific primers by reverse transcription polymerase chain reaction. The reactivity pattern of anti-HBs MAbs with different mutant forms of HBsAg was evaluated by enzyme-linked immunosorbent assay, and then the profile of antigen specificity and its association to VH/VL family expression was analyzed.
Results:
Twenty-three murine hybridomas producing anti-HBs MAbs were generated. Nucleotide sequence analysis revealed that heavy chains of these MAbs were encoded by IGHV genes from the HV1 (52%), HV6 (22%), HV5 (17%), and HV3 (9%) families in combination with IGHJ2 (57%), HJ1 (26%), and HJ4 (17%). Besides, 56% of MAbs used IGHD1 genes in their VDJ rearrangements. Concerning the IGKV gene, 26% and 22% of clones used KV4 and KV10 gene families, while the rest of the clones used KV8, KV6, KV1, KV12, and KV14 gene families. Besides, the IGKJ2 gene was the most represented KJ gene (43%). No association was found between the specificity pattern of MAbs to mutant forms of HBsAg with their preferential V, D, and J genes usage for most of MAbs.
Conclusion:
The data suggest that heavy chains of anti-HBs MAbs preferentially use genes derived from the IGHV1, IGHV6, IGHJ2, and IGHD1 families. In contrast to heavy chains, which predominantly use four families of IGHV genes, light chains use more diverse IGKV gene families.
Monoclonal antibodies (MAbs) were generated against subtypes (ad/ad/rw) of the human hepatitis B viral surface antigen (HBsAg). Among dozens of antibodies that were generated, the majority was shown to commonly react with various ad/ay subtypes of the S protein. Epitope(s) of these antibodies were grouped by various immunoassay methods, and at least four distinct epitope regions were identified. Some of these antibodies were selected to formulate sandwich enzyme immunoassays for quantitative determinations of HBsAg in reconstituted specimens. Epitope-defined monoclonal antibodies with high affinity and specificity might be suitable for formulations as vaccines (containing a mixture of humanized monoclonal antibodies) for passive immunization in humans for immunoprophylaxis of HBV infection.
This paper reviews a meeting at which basic pathophysiology of infections, mechanisms of action of hyperimmune products and pharmacokinetic and pharmacodynamic parameters, as well as currently available hyperimmunes and their potential new targets and uses, were discussed. A hyperimmune product was defined as either a monoclonal antibody or a polyclonal preparation enriched with antibody directed against one or more particular targets. A number of issues were emphasised, including: resistant bacterial pathogens, such as Staphylococcus aureus and Streptococcus pyogenes; the role of hyperimmune intravenous globulins in the prevention of sepsis in low birthweight infants; hepatitis B virus infection associated with liver transplantation; combination therapy; the potential role of hyperimmunes in the prevention and treatment of hepatitis C virus; and the use of immunoglobulins for the prophylaxis of Epstein-Barr virus-related lympho-proliferative disease. Routes of administration were also discussed. It was concluded that the development of hyperimmunes faces numerous obstacles. It was agreed that the use of hyperimmunes in clinical trials must be standardised; clinical trials must be large enough to have sufficient power to demonstrate efficacy with clear-cut end-points, and means need to be developed, in conjunction with regulatory agencies, for the feasible evaluation of combination products. However, progress in all these aspects will provide a wide range of hyperimmunes for future use.
Diagnosis of the hepatitis B Virus (HBV) is important for treatment and prevention of further spread of the virus. Today, detection of hepatitis B virus surface antigen (HBsAg) is the method of choice for the screening and initial diagnosis of HBV. Genetic diversity and discovery of HBV variants with mutations in the immunological dominant region of HBsAg caused none reactivity in some diagnostic assays. Characterization of anti-HBs monoclonal antibodies lead to discovery of a unique monoclonal antibody revealing the true topology of the small s-protein. The knowledge of the antibody recognition spectra, lead to the development of a diagnostics assay showing for the first time detection of all HBsAg mutants based on three different epitope regions. The three epitope regions should in theory enable detection of all known HBsAg mutants, which was proven after testing HBsAg mutants in direct comparison to other diagnostic assay lacking complete mutant detection.
The improved HBsAg screening assay permitted better and earlier diagnosis of HBV infections, leaving the diagnosed HBV patient for treatment. Today the main motivation for intervening in an on-going HBV infection is in case the liver is compromised. Depending on the status of the patient, antiviral therapy is started or in worse case when the liver stops to function, a liver transplantation is the final choice. Transplantation of the liver is preceded by antiviral therapy in combination with Hepatitis B immunoglobulin (HBIg) additions. In a quest for an alternative source for HBig we isolated and selected single-domain antibodies (VHHs) that recognize the major small s-protein of HBV. Testing of five VHH in an in-vitro neutralization experiment identified one VHH that could neutralize HBV comparable with good neutralizing anti-HBs reference antibodies. Pepscan analysis and amino acid substitution experiments suggested mutual recognition of the VHH of both the “a”-determinant and c-terminus of the s-protein, fixating the structure and preventing conformational changes needed during viral entry. Alternatively binding to a yet to be discovered HBV (co-) receptor is blocked by the VHH.
Ab initio modeling of the dimer “a”-determinant sequence (aa100-155) with VHH S5 revealed a tempting structure that could explain our and published observations. Mainly the location of the cysteine residues in the newly found structure was striking, showing possible formation of inter and intra chain disulfide bridges. Separate analysed c-terminal hydrophobic region (155-226) showed structural homology with Arfaptin 2, a BAR domain protein. The homologous structure predicted importance of the region giving curvature to the aggregated s-proteins in the HBsAg particles. We conclude from the modelling data that the protruding spikes are dimers of the “a”-determinant with peripheral stabilized c-terminal s-protein fragments on the 3- and 5-fold axis.
Virologic and clinical features of acute hepatitis B in the metropolitan area were analyzed with an emphasis on genotype A HBV. The proportion of patients infected with genotype A HBV was 31.8% between 1994 and 1997, 35.3% between 1998 and 2001, 41.5% between 2002 and 2005 and was increased to 73.0% between 2006 and 2008. The transmission route of genotype A HBV in recent years comprises sexual transmission from a steady Japanese heterosexual partner besides that from homosexual and unspecified heterosexual partners. One patient infected with genotype A HBV developed chronic hepatitis. Other two patients with a protracted course received Entecavir. Co-infection with human immunodeficiency virus was found in 14.3% of patients with genotype A HBV. Amino acid sequences in the "a-determinant region" were studied and demonstrated that one patient was infected with HBV which may escape vaccination. Education of people, surveillance of genotype A HBV and consideration of universal vaccination are important to prevent transmission of HBV, particularly with genotype A.
Current issues that are associated with the development of hepatitis B vaccine, combination vaccines, modes of administration, immunogenicity, and efficacy of different types of hepati- tis B vaccines are reviewed. Hepatitis B viral mutants can emerge as a result of either im- mune response or treatment options. Several studies are in progress on treatment of chronic hepatitis B infection by immunization with multiple antigenic components; DNA vaccines alone or with DNA encoded immunomodulatory cytokines; combination of vaccine with antivi- ral drugs and cytokines; and genetic manipulation of antigen presenting cells. Integrating hepatitis B vaccine doses into the global infant immunization program is not sufficient for hepatitis B virus (HBV) infection eradication. Implementing HBV schedule to high risk groups such as injection drug users, inmates of correctional centers, and persons at risk for sexually transmitted diseases, surveillance of hepatitis B infected subjects and refugees, access to immunization services and treatment is necessary. Further investigation is needed to assess factors that can impede an adequate antibody response, HBV variants, and the need for booster doses to preserve vaccine-induced immunity, vaccinating schedule for older children, evaluation of those vaccinated but in persistent contact in HBV-endemic areas.
A wide variety of commercial assays is available for the detection of HBsAg. Clearly, the sensitivity of an assay to detect a variant is dependent on the anti-HBs used. Thus, it is not surprising that there are examples of variants that cannot be detected by all assays. Accumulating data from Europe, Asia and Africa about HBsAg variants which are not recognized by either monoclonal or polyclonal antibodies specific for wild-type group 'a' determinant, but positive by DNA PCR in chronic patients and from vaccinated children are increasing. This would impose a challenge for public health issues of hepatitis B virus. In this review we tried to summarize the discrepancies between results of HBsAg assays and to explain some rationales for these inconsistencies.
Hepatitis B virus (HBV) remains a serious public health problem worldwide, accounting for high morbidity and mortality rates as well as significant personal, societal, and economic costs. Hepatitis B is a preventable disease; a safe and effective vaccine has been available for 30 years. The World Health Organization aims to control HBV worldwide by integrating HB vaccination into infant, possibly adolescent, and at-risk adult routine immunization programs. In recent years, a drastic reduction in the mortality and morbidity of chronic HBV, including hepatocellular carcinoma, has occurred, particularly in hyperendemic areas. In addition, a therapeutic vaccine that enhances patient immune response has been considered as a possible alternative to antiviral agents. However, mutant HBV may infect individuals who are anti-HBs positive after immunization (vaccine-escape) and/or fail for detection of HBsAg (diagnostic-escape), which may lead to transmission through donated blood or organs. This review attempts to summarize the prophylactic, therapeutic, and diagnostic concerns on HBV vaccines.
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.
The hepatitis B virus (HBV) polymerase gene completely overlaps with the envelope gene. In the present study we aimed to monitor the prevalence and pattern of the typical mutations for hepatitis B surface antigen (HBsAg) escape, and concomitantly nucleos(t)ide analog (NUC) resistance mutations, in Turkish patients undergoing different antiviral therapies and in treatment-naïve patients with chronic hepatitis B (CHB).
The investigation was undertaken between March 2007 and August 2009 and involved a total of 142 patients under NUC therapy (88 males; mean age 42 years (range 13-68); hepatitis B e antigen (HBeAg) negativity in 94 patients; HBV DNA median log 4.3 log(10) IU/ml (range 2.0->6.0); alanine aminotransferase (ALT) median level 76.1 IU/ml (range 12-1082)) and 185 treatment-naïve CHB patients (120 males; mean age 39 years (range 1-76 years); HBeAg negativity in 132 patients; HBV DNA median log 3.5 log(10) IU/ml (range 2.0-6.0); ALT median level 60.7 IU/l (range 8-874)).
The overall prevalence of typical HBsAg escape mutations found in the CHB patients was 8.3% (27/327). In the NUC therapy group the prevalence was 8.5% (12/142), with the following patterns: sY100C+sI110V, sL109I, sP120T, sP127T, sG130R+sG145X, sS132A+sY134N, sY134N+sG145R, sC137G, sD144E, sG145R. In the treatment-naïve group the prevalence was 8.1% (15/185), with the following patterns: sL109I, sI110V, sS117INST, sP120T, sP127T, sM133I, sC137L+sG145R, sS143L. However, NUC resistance mutations were found in 7.7% (11/142) of the patients on NUC therapy and 3.8% (7/185) of the treatment-naïve group patients. Interestingly, the treatment-naïve patients had preexisting drug resistance mutations related to lamivudine (rtL180M+rtM204I), adefovir (rtA181V, rtQ215S, rtI233V), entecavir (intermediate susceptibility with rtL180M+rtM204IHBV variant), telbivudine (rtL180M+rtM204I), and tenofovir (rtA194T).
The findings of this study show preexisting typical HBsAg escape and NUC resistance mutations are possible. The genetic arrangement of the HBV genome with polymerase and surface genes overlapping has substantial public health and diagnostic implications and relevance.
The impact of hepatitis B virus (HBV) genotypes on the sensitivity of surface antigen (HBsAg) detection assays has been poorly investigated. Here, plasmids carrying consensus or variant coding sequences for HBV surface proteins from genotypes A, D and F, were constructed. HBsAg levels were evaluated in medium and extracts of transfected CHO cells by a commercial polyclonal-based assay. We show that HBsAg detection values of consensus forms from genotypes D and F were, respectively, 37% and 30% lower than those obtained by genotype A. However, the presence of two single variations, T143M in genotype A, and T125M in genotype D, produced a decrease of 44% and an increase of 34%, respectively, on HBsAg mean values in comparison with their consensus forms. In conclusion, HBsAg detection levels varied among HBV genotypes. However, unique amino acid substitutions not linked to genotypes, such as T125M and T143M described here, should have more implications in HBV immunological diagnostics than the set of variations characteristic of each HBV genotype.
Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2007. A infecção pelo vírus da hepatite B é um dos maiores problemas de saúde pública no mundo, e a infecção viral mais importante entre profissionais de saúde. Diversas estratégias tem sido propotas paea controlar e prevenir esta infecção, mas para a adoção de medidas eficazes é necessário um amplo conhecimento sobre a epidemiologia da doença. Para estabelecer um panorama dessa infecção em nosso Estado, efetuou-se num estudo retrospectivo através da análise de exames executados pelo LACEN/FUNSAU/MS no període de 1997 a 2003, onde verificou-se que a ocorrência de 71,1% dos casos concentram-se nos municípios de Campo Grande, Dourados, Três Lagoas, Coronel Sapucaia, São Gabriel do Oeste, Naviraí, Ivinhema, Maracaju, Caaparó e Sonora. Através da análise da literatura disponível procurou-se a enfocar a transmissão da hepatite B como um risco ocupacional, em profissionais de saúde de diferentes categorias profissionais de saúe de diferentes categorias na rede pública de atenção primária. Participaram 332 profissionais de saúde que atuam nos municípios acima citados, com grande predominâcia do sexo feminino (86,7%) e idade média de 39 anos. Todos relataram já ter recebido pelo menos uma dose da vacina e 75%m apresentaram títulos de anti-HBsAg >10 UI/ml. Encontram-se marcadores sorológicos da infecção pelo HBV EM 11,1% das amostras analisadas com maior prevalência (51,3%) dos técnico/auxliares de enfermagem. A exposição à situação de risco para esta infecção foi identificada em 53,6% dos profissionais de saúde e o acidente ocupacional mais freqüente foi o ferimento por agulha. ______________________________________________________________________________________ ABSTRACT Hepatits B virus infection is one of the major world problems in public health and the most important viral infection among healthcareworkers. Various strategies have been proposed to control and prevent the infection, but for the adoption of effective control measures, a thorough understanding of the disease epidemiology is necessary. To estabilish this pathology scenery in Mato Grosso do Sul sdtate, a retropective study was carried out by the analysis of all requisitions for hepatitis B serology diagnosed by Central Public Health Laboratory , LACEN/FUNSAU/MS, from 1997 through 2003, when was founded that 71,1% of all cases were concentred in the municipalities, of Campo Grande, Dourados, Três Lagoas, Coronel Sapucaia, São Gabriel do oeste, Naviraí, Ivinhema, Maracaju, Caarapó and Sonora. The available literature was analysed, focusing the hepatitis B transmission, as an occupational risk in health care works from different professional categories who works in a public primary care network. A total of 332 healthcare workers, from the municipalities mencioned above participated, with grate female predominance (86,7%) and mean age of 39 years All healthcare workees declared that they have receive at least one dose of vacine, and 75,3% showed titer of anti-HSbAg > 10 UI/ml. Serelogical markers of HBV infection were found in 11,1% of the samples. The highest positive rate (51,3%) was found in nurse technicians and auxiliaries. The exposure risks to this infection was identified by 53,6% of the participants and the most frequent occupational acident was a needle stick.
A new antigen-antibody system associated with the hepatitis B virus and immunologically distinct from the HB surface, core, and e systems is reported. The new antigen, termed delta, was detected by direct immunofluorescence only in the liver cell nuclei of patients with HBsAg positive chronic liver disease. At present, the intrahepatic expression of HBcAg and delta antigen appears to be mutually exclusive. No ultrastructural aspect corresponding to the delta antigen could be identified under the electron microscope. delta antibody was found in the serum of chronic HBsAg carriers, with a higher prevalence in patients with liver damage. The nuclear fluorescence patterns of HBcAg and delta antigen were similar; it is only possible to discriminate between the two antigens by using the respective specific antisera.
A previous study (Carman, W. F., A. R. Zanetti, P. Karayiannis, J. A. Waters, G. Manzillo, E. Tanzi, A. J. Zuckerman, and H. C. Thomas. 1990. Lancet. 336:325-329) demonstrated a variant hepatitis B surface antigen (HBsAg) in a vaccinated child born to a hepatitis B virus-infected mother. A substitution of arginine for glycine at amino acid 145 in HBsAg was observed. In this study the effect of this substitution on the common "a" determinant of this protein, against which protective immunity is directed, is investigated. Using recombinant HBsAg with and without the amino acid substitution, the binding of monoclonal antibodies that recognize different epitopes of the "a" determinant, was shown to be destroyed by the presence of arginine at amino acid 145. In convalescent and vaccinee sera, antibody binding to HBsAg was not inhibited by the variant HBsAg. Immunization with the variant HBsAg, although eliciting a high titer antibody that recognized the variant, produced a low titer of antibody recognizing the native protein. Studies in mice demonstrate that the immunogenicity of the variant protein is also substantially altered. The data presented here demonstrate that this variant evades the known protective anti-HBs response and lends support to the suggestion that this mutation arose as the result of immune pressure.
Hepatitis delta virus (HDV) particles were produced in Huh7 human hepatoma cells by transfection with cloned hepatitis B virus (HBV) DNA and HDV cDNA. The particles were characterized by their buoyant density, the presence of encapsidated viral RNA, and their ability to infect primary cultures of chimpanzee hepatocytes. Successful infection was evidenced by the appearance of increasing amounts of intracellular HDV RNA after exposure to particles. Infection was prevented when particles were incubated with antibodies directed against synthetic peptides specific for epitopes of the pre-S1 or pre-S2 domains of the HBV envelope proteins before exposure to hepatocytes. These data demonstrate that HDV particles produced in vitro are infectious and indicate (i) that infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) that epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.
Expression of hepatitis B surface antigen (HBsAg), the major envelope protein of the virus, in the absence of other viral proteins leads to its secretion as oligomers in the form of disk-like or tubular lipoprotein particles. The observation that these lipoprotein particles are heavily disulphide crosslinked is paradoxical since HBsAg assembly is classically believed to occur in the ER, and hence in the presence of high levels of protein disulphide isomerase (PDI) which should resolve these higher intermolecular crosslinks. Indeed, incubation of mature, highly disulphide crosslinked HBsAg with recombinant PDI causes the disassembly of HBsAg to dimers. We have used antibodies against resident ER proteins in double immunofluorescence studies to study the stages of the conversion of the HBsAg from individual protein subunits to the secreted, crosslinked, oligomer. We show that HBsAg is rapidly sorted to a post-ER, pre-Golgi compartment which excludes PDI and other major soluble resident ER proteins although it overlaps with the distribution of rab2, an established marker of an intermediate compartment. Kinetic studies showed that disulphide-linked HBsAg dimers began to form during a short (2 min) pulse, increased in concentration to peak at 60 min, and then decreased as the dimers were crosslinked to form higher oligomers. These higher oligomers are the latest identifiable intracellular form of HBsAg before its secretion (t 1/2 = 2 h). Brefeldin A treatment does not alter the localization of HBsAg in this PDI excluding compartment, however, it blocks the formation of new oligomers causing the accumulation of dimeric HBsAg. Hence this oligomerization must occur in a pre-Golgi compartment. These data support a model in which rapid dimer formation, catalyzed by PDI, occurs in the ER, and is followed by transport of dimers to a pre-Golgi compartment where the absence of PDI and a different lumenal environment allow the assembly process to be completed.
Hepatitis delta virus (HDV) has an envelope composed of large-, middle-, and small-form hepatitis B surface antigens (HBsAgs) provided by the helper hepatitis B virus (HBV). In order to examine the roles of individual HBsAgs in HDV assembly, we constructed plasmids containing each specific HBsAg gene and then cotransfected each plasmid with HDV cDNA into a permissive human hepatoma cell line (HuH-7) to examine the effects on HDV production. Results indicated that the plasmids containing only the HBsAg genes were able to complement HDV cDNA as efficiently as the plasmid containing the complete HBV genome in generating HDV-like particles. Moreover, the small-form HBsAg alone was sufficient for HDV packaging. The particles produced from the cotransfection experiments have density and protein composition characteristics similar to those of naturally occurring HDV. With the electron microscope, they were identified as 36- to 38-nm-diameter particles. It was concluded that only the HBsAgs were able to help in the assembly of HDV-like particles.
Most of the lipid components of hepatitis B surface antigen (HBsAg) can be removed by treatment with the non-ionic non-denaturing detergent beta-D-octyl glucoside (OG) followed by centrifugation through caesium chloride linear density gradients (density 1.15-1.32 g/ml). The conformational changes induced by the elimination of lipids decreased the helical content of HBsAg proteins from 52 to 28% as indicated by c.d. techniques. Measurements of the extent of quenching of protein fluorescence by iodide showed that half of the tryptophan residues which are buried in the native structure of HBsAg particles are brought close to the surface of the molecule by such conformational changes. The antigenic activity, as measured by binding to polyclonal antibodies, was decreased upon removal of lipids. Moreover, the six different antigenic sites recognized by our panel of monoclonal antibodies decreased their capacity to bind to the corresponding antibody when lipids were removed. However, the extent of this decrease differed for the different antibodies. Thus the apparent dependence of antibody binding on the lipid content seemed to indicate a greater involvement of the lipid-protein interaction for some of the epitopes than for others.
Using a murine monoclonal antibody (RF-HBs-1) which has been shown to be capable of neutralizing both ad and ay subtypes of hepatitis B virus (HBV), we have devised a competitive inhibition assay to measure the presence of virus-neutralizing antibodies in the sera of patients who have recovered from acute type B hepatitis. The majority of patients have this antibody in their serum. We also show that this antibody inhibits the binding of polymerized human serum albumin (pHSA) to the pHSA receptor site of the HBV particle, which has been proposed as an important site for the entry of HBV into liver cells. We have demonstrated that the epitope recognized by this antibody is dependent on the linkage of 24,000 and 28,000 mol. wt. polypeptides via a disulphide bond. This conformational determinant in the coat of the virus which is part of or near to the pHSA binding site is important in evoking a virus-neutralizing response.
We have reported that clonal cells derived from Hep G2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete spherical and filamentous forms of hepatitis B surface antigen (HBsAg), core particles, and virions into the culture medium. Here we describe the development of typical hepatitis in two chimpanzees following intravenous inoculation with the medium in which the transfected cells had grown. The liver biopsies from these animals showed characteristic lesions in parenchyma and portal tracts, more conspicuous at an earlier time in the chimpanzee that had received a greater number of virions. The amount of HBsAg in the serum of one infected chimpanzee increased with time after the initial inoculation and then decreased concomitantly with the appearance of antibodies against HBsAg and core antigens. HBsAg remained detectable in the other animal throughout the course of the experiment. The levels of hepatitis B "e" antigen in both animals peaked at week 5, signifying the acute phase of the infection. The activities of serum enzymes that are markers for necroinflammation also increased. The hepatitis HBsAg subtype of the virions isolated from the patient whose DNA was cloned and then used for transfection of the Hep G2 cells was the same as that found in the chimpanzees. Furthermore, the restriction enzyme analysis of the viral DNA isolated from the chimpanzees was identical to the cloned DNA. Thus, HBV DNA-transfected Hep G2 cells can support the replication of virions that, in turn, produce hepatitis in chimpanzees.
Hepatitis B virus (HBV) DNA was extracted from sera of six carriers with hepatitis B e antigen as well as antibody to hepatitis B surface antigen and sequenced within the pre-S regions and the S gene. HBV DNA clones from five of these carriers had point mutations in the S gene, resulting in conversion from Ile-126 or Thr-126 of the wild-type virus to Ser-126 or Asn-126 in three carriers and conversion from Gly-145 to Arg-145 in three of them; clones with Asn-126 or Arg-145 were found in one carrier. All 12 clones from the other carrier had an insertion of 24 bp encoding an additional eight amino acids between Thr-123 and Cys-124. In addition, all or at least some of the HBV DNA clones from these carriers had in-phase deletions in the 5' terminus of the pre-S2 region. These results indicate that HBV escape mutants with mutations in the S gene affecting the expression of group-specific determinants would survive in some carriers after they seroconvert to antibody against surface antigen. Carriers with HBV escape mutants may transmit HBV either by donation of blood units without detectable surface antigen or through community-acquired infection, which would hardly be prevented by current hepatitis B immuneglobulin or vaccines.
Three assays, one based on monoclonal antibodies and the others on polyclonal antibodies, were employed to detect hepatitis B surface antigen (HBsAg)-reactive samples in both vaccinated and unvaccinated populations in areas of the world where hepatitis B virus (HBV) is endemic. Any discordant sera were tested by polymerase chain reaction (PCR) to confirm current infection, and sequence data were obtained from the DNA coding for the major hydrophilic region (MHR) of HBsAg of those samples positive for PCR. In all countries studied, samples that reacted in one HBsAg assay but not another were found. In the most extreme case, about 5% of viremic sera in Papua New Guinea were nonreactive in the monoclonal HBsAg assay; 9 of the 13 PCR-positive samples had novel or once-described variants, or a variant out of its usual genotype context. In South Africa, samples with sequences of subtype ayw2 reacted poorly, particularly in the polyclonal assay. Two had novel variants. In Sardinia, antibody to hepatitis B core antigen (anti-HBc) was analyzed as a marker of infection. A significant proportion of anti-HBc-positive, but monoclonal HBsAg-negative, vaccinees and unvaccinated persons were found to be PCR positive, as were some individuals without any markers of hepatitis B virus infection. Five more novel variants were found in these groups. There are implications for the design of HBsAg assays, which may have to be modified according to local sequence variability. Not all discordant samples were explained by variants, indicating that assay sensitivity is fundamental to diagnostic efficacy. Overall, this study defined 16 novel variants and 2 new potential epitope clusters. (Hepatology 1997 Dec;26(6):1658-66)
Mutational changes in the pre-S region of hepatitis B virus (HBV) were analyzed in 20 patients who experienced HBV reinfection after orthotopic liver transplantation (OLT). HBV DNA was extracted from patient sera before and after OLT. The pre-S sequence was amplified via polymerase chain reaction, subcloned, sequenced, and analyzed. In 18 of 20 patients, mutational changes were found in the pre-S region pre- or post-OLT; 11 showed point mutations (1-10) and 7 cases major changes (insertions/deletions). For the point mutations, there was no trend in the selection of wild-type (wt) HBV before or after OLT in the pre-S region. Additional HBV reinfection during hepatitis B surface antigen antibody (anti-HBS) administration had no influence on selection pressure in the pre-S region. In contrast, insertions/deletions were more frequently found before OLT. In the 7 patients with deletions/insertions, changes in the hepatocyte attachment site were not seen after OLT. Interestingly, the only patient with changes in a major virus population after OLT had changes in the CCAAT-box of the S-promoter. As shown by gel shift analysis, this mutation was associated with loss of specific binding to this element and thus probably led to dysregulation of S-gene transcription. Major changes in the pre-S genome are mainly seen before OLT, and HBV reinfection does occur with the intact hepatocyte attachment sites after OLT. Anti-HBs (hepatitis B immune globulin [HBIg]) creates no selection pressure on the pre-S region. The mutation in the CCAAT-box of the S-promoter potentially leads to its dysregulation and may be associated with the occurrence of fibrosing cholestatic hepatitis after OLT. (Hepatology 1996 Sep;24(3):482-8)
To identify the mechanisms of viral persistence in patients with chronic hepatitis B after the acquisition of anti-hepatitis B surface antigen antibodies (antiHBs), we serially analyzed the nucleotide sequence of the envelope region in a cohort of infected patients. Four patients with histological diagnoses of chronic hepatitis B who had at least 5 years of observance by our hospital staff were studied. All but one showed normalization of serum alanine aminotransferase (ALT) concentration after clearance of the hepatitis B surface of antigen (HBsAg) and the appearance of anti-HBs. Hepatitis B virus (HBV) DNA was still detectable by polymerase chain reaction (PCR) amplification assay in serum specimens from two patients, even in the presence of circulating anti-HBs. The envelope gene was amplified by PCR in serum samples obtained both before and after seroconversion, and direct cycle sequencing of the PCR products was performed. A mutation resulting in a premature stop codon was found in the pre-S1 region of one patient just prior to clearance of HBsAg. Two years later, the stop codon was converted to a leucine codon and three mutations developed in the "a" loop. In the other patient, 16 amino acids had been deleted between amino acids 8 and 23 in the pre-S2 region before clearance of HBsAg. After the appearance of circulating anti-HBs, the pre-S2 gene reverted to the wild type but three additional mutations appeared inside the "a" loop. These results suggest that HBV mutates when HBsAg is cleared, which may contribute to viral persistence due to an evasion of the host immune surveillance. (Hepatology 1996 Mar;23(3):389-95)
Serum hepatitis B virus (HBV) DNA from 4 infants with fulminant hepatitis B, 3 infants with acute self-limited hepatitis B, and 15 infants with chronic HBV infection were amplified by polymerase chain reaction followed by direct sequencing of the region of HBV genome encoding the major antigenic epitopes of hepatitis B surface antigen (HBsAg). All infants were born to carrier mothers and administered immunoprophylaxis from birth. Serum HBV DNA from 13 carrier children born to carrier mothers who did not receive immunoprophylaxis and had comparable length of infection were studied as controls. An S mutant (residue 126, Thr to Ala) initially found in an infant with fulminant hepatitis was replaced by another S mutant (residue 145, Gly to Arg) 4 days later. In a girl with chronic hepatitis B, Ala-126 variant and Arg-145 variant were found at 17 and 25 months of age, respectively. The Arg-145 variant persisted for 8 years in an asymptomatic male carrier and for 1 year in an infant with chronic hepatitis B. The Ala-126 variant persisted for 11 years in one child who had an early loss of hepatitis B e antigen. In the majority of the infants' mothers, corresponding mutations in HBsAg were not detected in serum by direct sequencing. The S mutants detected in three carrier infants were not found in their mothers' serum after cloning and sequencing of 10 DNA clones from each maternal sample. None of the 13 control patients had detectable S mutants. These results suggest that S variants emerge or are selected under the immune pressure generated by the host or by administration of hepatitis B immune globulin and hepatitis B vaccination. An S mutant (residue 129, Gln to Arg) found in one mother-infant pair suggested a direct maternal-infant transmission, resulting in immunoprophylaxis failure. None of the family members of children infected with Arg-145 variant had the same variant infection, implying this variant's low transmissability. (Hepatology 1997 Sep;26(3):786-91)
Hepatitis B virus (HBV) replicates via an intermediate RNA step. High frequency of polymerase errors with additional selection pressure leads to mutations in the HBV genome. We investigated the number, type, and antigenic effects of mutations in the coding region of the HBV surface antigen in eight patients who underwent orthotopic liver transplantation (OLT) for HBV-related end-stage liver disease and were experiencing infection of the graft and who received hepatitis B surface antigen antibody (anti-HBs) prophylaxis (hepatitis B immune globulin [HBIG]) after OLT. Controls were chronic HBV patients who underwent kidney transplantation and received the same immunosuppressive regime but no HBIG. The S-gene was amplified from serum before and after transplantation, sequenced, and changes in the genome were analyzed. In the five patients who experienced reinfection while receiving anti-HBs, clear mutations occurred in the S-gene. In the patient who did not receive HBIG and those who experienced reinfection only after termination of HBIG, no mutations were found in the S-gene. In the kidney recipients, mutations in the S-gene occurred in only one of eight patients. Because the a determinant contains neutralizing epitopes, this region was chosen for antibody binding to quantify antigenic effects of the mutations. The two patients who selected mutations in the a determinant and became reinfected while receiving HBIG had reduced antibody binding after OLT. Our results suggest that HBIG after OLT imposes a selection pressure on the S-gene, and that mutations are one mechanism for reinfection while receiving HBIG. (Hepatology 1996 Sep;24(3):489-93)
Hepatitis B virus (HBV) DNA was extracted from sera of six carriers with hepatitis B e antigen as well as antibody to hepatitis B surface antigen and sequenced within the pre-S regions and the S gene. HBV DNA clones from five of these carriers had point mutations in the S gene, resulting in conversion from Ile-126 or Thr-126 of the wild-type virus to Ser-126 or Asn-126 in three carriers and conversion from Gly-145 to Arg-145 in three of them; clones with Asn-126 or Arg-145 were found in one carrier. All 12 clones from the other carrier had an insertion of 24 bp encoding an additional eight amino acids between Thr-123 and Cys-124. In addition, all or at least some of the HBV DNA clones from these carriers had in-phase deletions in the 5' terminus of the pre-S2 region. These results indicate that HBV escape mutants with mutations in the S gene affecting the expression of group-specific determinants would survive in some carriers after they seroconvert to antibody against surface antigen. Carriers with HBV escape mutants may transmit HBV either by donation of blood units without detectable surface antigen or through community-acquired infection, which would hardly be prevented by current hepatitis B immuneglobulin or vaccines.
The formation of disulfide bonds between cysteine residues stabilizes protein structures, and thus, plays critical roles in protein folding, function, and evolution. Essentially, knowing the disulfide connectivity pattern is helpful in predicting the three-dimensional structures of proteins due to its capability to restrain the flexibility of protein structures and to reduce the search in the conformational space. As a result, disulfide connectivity prediction from protein sequences has become more attractive and feasible with the rapid accumulation of complete genome sequences and the advances in bioinformatics and machine learning methods. In this review, we will describe the methods associated with disulfide bonding state and connectivity prediction in detail. In addition, the main issues and limitations will also be presented along with the methods discussed. Finally, the practical applications and some possible future directions of disulfide connectivity predictions will also be mentioned.
Peripheral blood mononuclear cells from a donor with a high litre of anti-hepatitis B surface (HBs) antibodies were fused with a cell line that was positive for Epstein-Barr virus nuclear antigen and sensitive to hypoxanthine-aminopterine-thymidine. A cell line was established that produces a monoclonal IgG1λ anti-HBs antibody. Afterwards, it appeared that the anti-HBs antibody-producing cell line had arisen from Epstein-Barr virus transformation of the donor B cells. The cell line is capable of producing up to 60 μg/ml of the monoclonal antibody, which has a high avidity for HBs antigen (Ag) and recognizes both ad and ay subtypes. The antibody is useful as a reagent for the detection of HBsAg in human serum. Over 1000 patient sera have been tested with a conventional third-generation assay in parallel, and only a single discrepant serum was found.
The general applicability of the new peptide immobilization strategy in which the peptide of interest is N-terminally extended with an acetyl-thio-acetyl group or (poly)-Lys extension during synthesis, has been demonstrated in epitope-mapping experiments and serodiagnosis. Ala-scanning experiments and minimal epitope determination showed that the antigenicity of Ata-extended peptides derived from the human chorionic gonadotropin (hCG) and hepatitis B virus (HBV) amino acid sequence, was superior to the free parent peptides. Further, it could be shown that the choice of the epitope-mapping procedure (peptide in solution or immobilized on a solid support) may lead to a different perception of which residues constitute the epitope. In addition, a time-consuming conjugation process could be circumvented since the ELISA reactivity of BSA-conjugates was comparable to that of Ata-extended peptides. In the serodiagnosis using sera from various HIV-positive individuals, the lysyl-peptide showed a signal/noise ratio 10 times higher than the parent peptide, indicating that sensitivity increased as a result of this N-terminal lysyl tail. In all experiments we observed that antibody detection could be performed at roughly 10 times lower amounts of peptide when N-terminally linked to an Ata-group or lysyl-extension compared to the free parent peptide or the BSA-conjugated equivalent.
After cell hijacking and intracellular amplification, non-lytic enveloped viruses are usually released from the infected cell by budding across internal membranes or through the plasma membrane. The enveloped human hepatitis B virus (HBV) is an example of virus using an intracellular compartment to form new virions. Four decades after its discovery, HBV is still the primary cause of death by cancer due to a viral infection worldwide. Despite numerous studies on HBV genome replication little is known about its morphogenesis process. In addition to viral neogenesis, the HBV envelope proteins have the capability without any other viral component to form empty subviral envelope particles (SVPs), which are secreted into the blood of infected patients. A better knowledge of this process may be critical for future antiviral strategies. Previous studies have speculated that the morphogenesis of HBV and its SVPs occur through the same mechanisms. However, recent data clearly suggest that two different processes, including constitutive Golgi pathway or cellular machinery that generates internal vesicles of multivesicular bodies (MVB), independently form these two viral entities.
Hepatitis B virus consists of an icosahedral core containing the double-stranded DNA genome, enveloped by a membrane with embedded surface proteins. The crystal structure of the core protein has been solved but little information about the structure of the surface proteins has so far been available. There are three sizes of surface protein, small (S), medium (M) and large (L), which form disulfide-bonded homo- and heterodimers. The three proteins, expressed from different start sites in the coding sequence, share the common C-terminal S region; the M protein contains an additional preS2 sequence N-terminal to S, and the L protein a further preS1 sequence N-terminal to M. In infected individuals, the surface proteins are produced in huge excess over the amount needed for viral envelopment and are secreted as a heterogeneous mixture of isometric and tubular subviral particles. We have used electron cryomicroscopy to study tubular particles extracted from human serum. Helical Fourier-Bessel analysis was used to calculate a low-resolution map, although it showed that the tubes were quite disordered. From the symmetry derived from this analysis, we used single-particle methods to improve the resolution. We found that the tubes had a diameter of approximately 250 A, with spike-like features projecting from the membrane. In the plane of the membrane the proteins appear to be close packed. We propose a model for the packing arrangement of surface protein dimers in the tubes.
Unlabelled:
Hepatitis B virus (HBV) infections cause 500,000 to 700,000 deaths per year as a consequence of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Efficient and safe antivirals to treat chronically infected patients and consequently to prevent development of hepatocellular carcinoma are still awaited. We isolated five single-domain antibodies (VHHs) that recognize the most abundant envelope protein (S) of HBV. VHHs, when expressed and retained in the endoplasmic reticulum as intrabodies, reduced levels of secreted hepatitis B surface antigen (HBsAg) particles in a cellular HBV model. In a hydrodynamics-based HBV mouse model, these intrabodies caused a marked reduction in HBsAg concentrations and a 10- to >100-fold reduction in the concentration of HBV virions in plasma.
Conclusion:
VHHs potently inhibited secretion of HBV virions in vivo, showing that this approach might be useful in the treatment of HBV. To our knowledge, this is the first report of intrabody-mediated inhibition of viral secretion in mammals.
We have found previously that hybrid 22-nm HBsAg particles can be created by insertion of short antigenic sequences into the HBV major envelope protein. We have now performed a detailed deletion mutagenesis of the S gene of HBV encoding HBsAg. Deletion of the 51 C-terminal amino acids including most of the third and all of the fourth hydrophobic domain of the S protein did not affect particle assembly and secretion. However, secretion of 22-nm particles was abolished by minor deletions in the N-terminal region. Insertion and deletion/substitution mutants carrying a poliovirus epitope at the N-terminus and the preS1 region at the C-terminus have been characterized.
Vaccination with hepatitis B surface antigen (HBsAg) has shown that antibody directed against the common 'a' determinant of this antigen is protective against infection with hepatitis B virus (HBV). In this study the antigenic epitopes of the 'a' determinant have been analysed by competitive inhibition assays and by binding studies to synthetic peptides using a panel of monoclonal antibodies prepared against HBsAg, all of which are shown to recognise the common group determinant. One murine monoclonal antibody used in this study, RFHBs1, has been shown previously to block infectivity of HBV in susceptible chimpanzees ((1983) J. Med. Virol. 16, 89-95). This antibody bound to a cyclical synthetic peptide analogue of amino acids 124 to 137 of the major HBsAg polypeptide.
A variant of hepatitis B virus (HBV) having a specific mutation within the S gene has been found to infect vaccinees. To know whether similar variants were involved in Japan, we analyzed two cases of maternal transmission of HBV in infants immunized with hepatitis B immune globulin and hepatitis B vaccine. DNA clones of HBV S genes were propagated from patients and family members and sequenced. In one family, the DNA clones from the baby patient had a Gly-to-Arg mutation at the 145th codon of the S gene, whereas those from her mother had no such mutations. In the other family, all the DNA clones obtained from the two infected children had the 145th codon intact, but they had a missense mutation at the 126th codon of the S gene, causing an amino acid substitution of Asn for Thr or Ile. This same mutation was observed in 12 of 17 clones of DNA obtained from their mother. In comparison with the wild type HBV-derived hepatitis B surface antigen, the two types of S gene mutations, either at the 145th or the 126th codon, were associated with a significant decrease in the antigenicity of some determinants on the hepatitis B surface antigen, measured by MAb. Amino acid substitution at these sites, therefore, would have induced the escape from conventional vaccines that were S gene products of wild type HBV and also from hepatitis B immune globulin, whose main components were probably also antibodies against the S gene products expressed by wild type HBV.
Amino acid residues 101 to 180 of hepatitis B surface antigen (HBsAg) were predicted by sequencing the corresponding part of the S gene of hepatitis B virus (HBV) DNA in 46 HBsAg-positive sera, which had been subtyped by immunodiffusion with respect to d/y, w/r, w1 to w4 and q. The sequences of the nine different HBV serotypes defined by these specificities were found to be homogeneous proving that they represent consistent variations of HBV at the genomic level. Residue 127 was found to be important as were Pro, Thr and Leu for w1/w2, w3 and w4, respectively. Five residues were found to differ between ayw1 and ayw2. These were at positions 134 (Phe instead of Tyr), 143 (Thr instead of Ser), 159 (Ala instead of Gly), 161 (Tyr instead of Phe) and 168 (Val instead of Ala). However, all these residues were shared by ayw1 and adw2, implying that Arg122 was also important for w1 expression. All genomes expressing r, apart from one ayr strain, had an Ile126, which might explain the pseudo-allelism of w1 to w4 in relation to r, since this substitution might influence the w epitope. There were two regions where adw4q- and adrq- differed from all the q+ subtypes. These were located at residues 158 and 159, and at residues 177 and 178, where both the q- subtypes had amino acid substitutions in adjacent positions. The mapping of the epitopes defining these antigenic specificities will help to link information on the world-wide distribution of HBsAg subtypes to future molecular epidemiology with regard to HBV.
A model of hepatitis B surface antigen has been derived, based on extensive sequence analysis and biochemical data. The surface antigen sequences of the human, woodchuck, ground squirrel and duck hepadnaviruses were examined using hydrophobicity, hydrophobic moments, flexibility and secondary structure prediction. The helix phase diagram, which is a modified version of Eisenberg's hydrophobic moment plots and which specifically addresses the problem of transmembrane helices, was used to examine the predicted helices. In this model four transmembrane helices are predicted. The N and C termini and the second hydrophilic region, which bears the major B-cell antigenic determinants, are external. It is suggested that the transmembrane helices may pack to form a channel through the membrane and may also be involved in the mechanisms of cell entry. A significant difference between the duck hepadnavirus and the mammalian HBsAg sequences was found, hence care must be taken when extrapolating data between the duck and the human surface antigen.
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.
A gene region encoding a segment of the major surface protein, HBsAg, of hepatitis B virus was analyzed from serum samples after orthotopic liver transplantation of three hepatitis B virus chronic carrier patients treated with a human anti-hepatitis B virus monoclonal antibody (SDZ OST 577). Each of these three patients became HBsAg negative after transplantation and therapy with the human anti-hepatitis B virus monoclonal antibody but returned to HBsAg positivity (first detected 143,251 and 252 days after the transplantation). Polymerase chain reaction DNA amplification was performed on DNA from serum samples showing low levels of recurrent HBsAg and reduced antigen reactivity with SDZ OST 577 antibody. Polymerase chain reaction DNA included a 230-bp highly conserved, major S gene region that was cloned into M13 bacteriophage; analysis of this DNA segment provided a consensus of DNA sequences for the serum samples exhibiting altered reactivity with the therapeutic monoclonal. Analysis of independent DNA clones from serum samples of patients exhibiting low but detectable recurrent serum levels of posttherapy HBsAg revealed the presence of S protein variant sequences when compared with polymerase chain reaction DNA derived from the original infected liver or pretherapy serum HBsAg. Genetic variation was predominant in a highly conserved peptide domain that has previously been implicated in antibody binding and neutralizing antibody epitopes. In independent patients infected with either adw or ayw hepatitis B virus subtypes, single nucleotide changes resulted in one to two amino acid differences for each variant allele (residues 124, 129, 131, 137, 140 and/or 145) when compared with pretherapy viral DNA. Administration of serum containing one of these variant viruses to a single hepatitis B-naive chimpanzee resulted in subclinical hepatitis and detectable levels of circulating anti-HBs and anti-HBc antibodies 49 and 70 days after virus administration, respectively. Hepatitis B virus DNA was recovered on liver biopsy between 6 and 8 wk after inoculation, although the animal remained persistently seronegative for HBsAg. DNA sequence analysis of both primate and patient liver hepatitis B virus confirmed the presence of the DNA encoding the S protein variant and associates this DNA with the predominant hepatotropic virus in liver infection.
The surface (S) genes of 12 hepatitis B viruses (HBVs) encoding nine different serotypes of hepatitis B surface antigen (HBsAg) were amplified by the polymerase chain reaction and sequenced. These represented the eight strains of HBV, P1 to P8, defined at an international workshop on HBsAg subtypes in Paris in 1975, and the adrq- subtype. The S genes from additional HBV strains, one ayw4, one adw4 and one ayw1, of sub-Saharan African origin, were also sequenced. The relationship of these 12 new S gene sequences to those of the 20 published previously was investigated by constructing a phylogenetic tree, which confirmed a previous classification into four groups, designated A to D, based on 18 complete HBV genomes. When relating our sequenced S genes to these genomic groups, ayw1 of African origin and P6 (adw2) were both allocated to group A, the reference P1 (ayw1 of Vietnamese origin) was allocated to group B, P5 (ayr), P8 (adr) and adrq- were all related to group C, and P2 (ayw2) and P3 (ayw3) could both be allocated to group D. Interestingly, the S genes of w4 serotype viruses, i.e. P4 (ayw4) and P7 (adw4q-), differed by 4% or more from both previous groups and from each other, suggesting their classification into two new groups, for which the designations E and F are proposed. Genomes specifying ayw were also found in groups A and B; previously sequenced genomes specifying the ayw subtype have all been confined to group D. There were indications that the epitope for subdeterminants of w resided at amino acid positions 125 to 127. Thus, at positions 125 and 127, ayw1, ayw2 and adw2 had T and P residues, respectively, whereas M and T residues were at the corresponding positions of ayw3. Both ayw4 and adw4 had L at residue 127, and all strains expressing r, apart from P5, had an I instead of a T residue at position 126.
Spontaneous intestinal and intra-abdominal bleeding was observed in a high percentage of newborn transgenic mice carrying the murine urokinase-type plasminogen activator (uPA) gene linked to the albumin enhancer/promoter. These hemorrhagic events were directly related to transgene expression in the liver and the development of high plasma uPA levels. Two lines were established from surviving founder mice that displayed multigenerational transmission of the bleeding phenotype. Fatal hemorrhaging developed between 3 and 84 hr after birth in about half of the transgenic offspring of these lines; transgenic pups that did not bleed nevertheless passed the phenotype to their young. The phenotypic variability could not be explained by differences in transgene expression. All transgenic neonates were severely hypofibrinogenemic and displayed loss of clotting function that extended beyond the risk period for bleeding. These mice provide a means of studying the pathophysiology of plasminogen hyperactivation and evaluating therapeutic protocols designed to prevent bleeding.
In southern Italy, 44 contacts of hepatitis B virus carriers, including infants of carrier mothers, became HBsAg positive despite passive and active immunisation according to standard protocols. In 32 of these vaccinees infection was confirmed by the presence of additional markers of viral replication. In 1 infant, serious disease occurred. The virus from this patient is an escape mutant with a different sequence from that of the isolate from the mother. A point mutation from guanosine to adenosine at nucleotide position 587 resulted in an aminoacid substitution from glycine to arginine in the highly antigenic a determinant of HBsAg. This mutation is stable: it is present in an isolate from the child 5 years later. In some of these patients, including this child, the a determinant, to which a large part of the vaccine-induced immunity is directed, has been partly lost. Binding to HBsAg of a monoclonal antibody, previously mapped to the region of the mutation, was reduced in the child relative to that of the mother.
Two determinants of hepatitis B surface Ag (HBsAg), identified by mAb raised against polypeptide components, were characterized immunochemically. One was expressed on HBsAg irrespective of the four major subtypes, i.e., adw, adr, ayw, and ayr, whereas the other was subtypic but not identical to any of d, y, w, and r determinants. The common determinant was generated by a synthetic pentadecapeptide with a sequence of Thr-Thr-Ser-Thr-Gly-Pro-Cys-Lys-Thr-Cys-Thr-Ile-Pro-Ala-Gln representing amino acids 115-129 of the S gene product, and detected invariably in 366 HBsAg samples in sera from asymptomatic carriers in Japan. The activity of the S gene product, as well as the peptide (115-129), to bind with the mAb was not affected by alkylation alone, but was completely lost after reductive alkylation. The antigenic activity was lost when the S gene product was severed between Lys122 and Thr123 by trypsin. A microconformation maintained by the -Cys121-Cys124 bond, therefore, would be required for the common determinant. The other mAb identified an epitope of HBsAg that was mimicked by a synthetic tetradecapeptide with a sequence of Thr-Cys-Thr-Ile-Pro-Ala-Gln-Gly-Thr-Ser-Met-Phe-Pro-Ser, representing amino acids 123-136 of the S gene product. Among 16 HBsAg samples with known S gene sequences, 5 with Ile126 possessed this subtypic determinant, but the remaining 11 with Thr126 did not. The 5 hepatitis B virus genomes encoding the subtypic determinant differed less than 5.6% from each other in the entire nucleotide sequence, but by 8.0% or more from any of the other 11 genomes without the capacity to encode it.
We investigated the case of a child who was vaccinated at birth against hepatitis B and was also given hepatitis B immune globulin, but who nevertheless later became infected with the virus. Hepatitis B virus-specific deoxyribonucleic acid covering the region of the genome encoding the predominant a epitope of hepatitis B surface antigen was amplified using the polymerase chain reaction and the nucleotide sequence determined. We present evidence for the independent emergence of an escape mutant, similar to that previously identified in Italy, where a substitution of arginine for glycine has occurred in the second loop of the a epitope.
Although HBV has a circular DNA genome that is partially double stranded, it replicates by means of an RNA intermediate. The process is catalyzed by a translation product of the polymerase open reading frame that has reverse transcriptase activity. The enzyme is found in association with the virion and achieves a high rate of nucleotide misincorporation during transcription because such enzymes lack proofreading activity. The virus is remarkable for its efficient use of nucleic acid because the genome is only 3.2 kb long and yet it encodes four groups of proteins and their regulatory elements (1). This is achieved in some regions by reading the sequence through different frames to direct the synthesis of distinct proteins from the same genetic material. Only a few of the mutations that occur during the normal replication cycle therefore permit the entry of a new virus to the pool for natural selection. Nevertheless, patients with chronic hepatitis have been shown to have viruses with different sequences cocirculating, and some regions of the genome are poorly conserved between different isolates (2).
The existence of patients with evidence of HBV replication but no evidence of liver disease, both in the early phase of acute infection (3) and in some chronic infections (4), has led to the suggestion that the virus is not directly cytopathic but that it is the immune response to infected liver cells that causes the hepatitis (5). The development of polymerase chain reaction (PCR) sequencing techniques has allowed us to determine whether sequence variation occurs under host selection and offers an explanation for the variable outcome of HBV infection.
Cells infected with hepatitis B virus produce both virions and 20-nm subviral (surface antigen or HBsAg) particles; the latter are composed of viral envelope proteins and host-derived lipid. Although hepatitis B virus encodes three envelope proteins (L, M, and S), all of the information required to produce an HBsAg particle resides within the S protein. This polypeptide spans the bilayer at least twice and contains three hydrophobic regions, two of which are known to harbor topogenic signal sequences that direct this transmembrane orientation. We have examined the effects of mutations in these and other regions of the S protein on particle assembly and export. Lesions in the N terminal signal sequence (signal I) can still insert into the endoplasmic reticulum bilayer but do not participate in any of the subsequent steps in assembly. Deletion of the major internal signal (signal II) completely destabilizes the chain. Deletion of the C-terminal hydrophobic domain results in a stable, glycosylated, but nonsecreted chain. However, when coexpressed with wild-type S protein this mutant polypeptide can be incorporated into particles and secreted, indicating that the chain is still competent for some of the distal steps in particle assembly. The correct transmembrane disposition of the N terminus of the molecule is important for particle formation: addition of a heterologous (globin) domain to this region impairs secretion, but the defect can be corrected by provision of an N-terminal signal sequence that restores the proper topology of this region. The resulting chimeric chain is assembled into subviral particles that are secreted with normal efficiency.
We have shown, by analyzing serial serum samples from a chronic hepatitis B virus (HBV) carrier, the emergence of HBV DNA molecules with nucleotide rearrangements in the pre-S/S and pre-C/C genes. Serum samples were obtained at four different times (1983, 1985, 1988, and 1989) from an HBsAg- and HBeAg-positive carrier with chronic hepatitis. The polymerase chain reaction was used to amplify the pre-S/S and pre-C/C genes. The amplified products were cloned, and 8 to 10 independent clones were sequenced. In 1983 and 1985 only one type of HBV DNA molecule was observed. Nucleotide divergence relative to the adw2 subtype was 4.7, 7.2, and 1.6%, for the pre-S1, pre-S2, and S regions, respectively, and 2.2 and 3.9% for the pre-C and C regions, respectively. In 1988 and 1989, HBV DNA forms with marked rearrangements of both the pre-S/S and pre-C/C regions were evidenced. In the pre-S/S region, they comprised two distinct HBV DNA molecules. The first showed nucleotide divergence of 20.4, 14.8, and 3.3% for the pre-S1, pre-S2, and S regions when compared with the adw2 sequence. In addition, nucleotide deletions in the pre-S1 region led to the appearance of a stop codon. The second was created by recombination between the original and mutated HBV DNA. In the pre-C/C region, the mutated viral DNA showed 11.7% divergence when compared with the adw2 sequence. A point mutation led to the creation of a stop codon in the pre-C region, together with an insertion of 36 nucleic acids in the core gene. Most of this DNA insertion was identical to that reported in an independent HBV isolate but showed no significant homology with known sequences. Semiquantitative estimation of the proportion of wild-type and mutated HBV DNA molecules showed a marked increase in the mutated forms during the period of follow-up. Sucrose gradient analysis indicated that the defective HBV DNA molecules were present in circulating virions. Western immunoblot analysis showed the appearance of modified translation products. Our findings thus indicate the emergence of and gradual takeover by mutated HBV DNA forms during the HBV chronic carrier state. The rearrangements we observed in the pre-S/S and pre-C/C genes might lead to changes in the immunogenicity of the viral particles and thus affect the clearance of the virus by the immune system.
An in vitro system for production of hepatitis B virus (HBV) was established by infection of human hepatoma (HepG2) cells. HBV particles obtained from the serum of a chronic hepatitis B surface antigen (subtype ad) carrier were used to inoculate HepG2 cells. HBV envelope and core proteins were synthesized de novo by the infected cells and secreted into the medium 3 to 6 days postinfection. Viral covalently closed circular DNA, the putative template for viral RNA transcription, accumulated in the cells with increasing time postinfection. The HBV-infected HepG2 cells were maintained for several months (HepG2-BV cell line) and continued producing viral antigens. Both HBV DNA replicative intermediates and major HBV transcripts were identified in HepG2-BV cells. Complete HBV particles, which contain HBV DNA and DNA polymerase activity and express the three antigenic specificities of the envelope (hepatitis B surface antigen, pre-S2, and pre-S1), were released into the culture supernatant. Thus, successful in vitro infection of transformed human hepatocytes raising stable HBV-producing cells was achieved for the first time. This strongly suggests that HepG2 cells have a receptor(s) for virus attachment and penetration. Such a system represents a significant advance for the study of HBV-target cell interactions as the early events of HBV infection.
A likely mechanism of the strong hepatotrophism of the hepatitis B virus is the presence of specific receptors for the surface antigen of hepatitis B virus on hepatocyte membranes. To examine this hypothesis, we have performed binding studies using recombinant large (preS1 + preS2 + S) and major (S) proteins with adult human hepatocytes, rat hepatocytes, human fibroblasts, human peripheral blood mononuclear cells and plasma membranes derived from these cell types. We found that major HBsAg was able to bind specifically to human hepatocytes, human fibroblasts and human blood mononuclear cells. This binding could be inhibited by recombinant middle (preS2 + S) protein but not by the recombinant large protein. No binding could be demonstrated between large HBsAg and human hepatocytes. However, large protein bound specifically to plasma membranes derived from human liver tissue, human fibroblasts and HepG2A16 cells. This binding could be partially inhibited by the major protein and by a synthetic preS1 peptide but not by a synthetic preS2 peptide. These results support the assumption that hepatitis B virus absorption and penetration into human hepatocytes is mediated by specific receptors recognizing an amino acid sequence in the S-region. This recognition site is not displayed by the recombinant large protein. However, the large protein is recognized by its preS1 region and by a second binding site in the S-region by a receptor molecule, located on the inner surface of the plasma membranes or intracellular membranes of human hepatocytes and of some other cell types derived from human tissue.
Two study chimpanzees were inoculated intravenously with approximately 1,000 chimpanzee infectious doses of hepatitis B virus (HBV), one with subtype adr and one with subtype ayw, each previously incubated with 0.1 ml of a murine monoclonal antibody (IgG 1(K) class) directed against a single epitope on hepatitis B surface antigen common to most or all HBV. Two control chimpanzees received identical doses of HBV not incubated with the murine anti‐HBs.
Neither study chimpanzee developed HBV infection during 12 months of follow‐up as judged by normal serum aminotransferase activity, normal liver biopsies, and negative serological tests for HBV‐associated antigens and antibodies. In contrast, both control chimpanzees became infected by HBV as evidenced by elevated serum aminotransferase activity, liver biopsy changes characteristic of viral hepatitis, and the appearance of hepatitis B surface antigen (HBsAg) in their sera. Both study chimpanzees were shown to be fully susceptible to infection with these same HBV inocula when challenged 15 months after the initial inoculations at a time when passively administered anti‐HBs was no longer detectable. Prior to challenge with HBV, one of the two study chimpanzees received a second injection of the same volume of the murine monoclonal anti‐HBs. The survival of this anti‐HBs in serum was reduced from six weeks (after the initial injection) to approximately two weeks.
This study documents the biological efficacy of a murine monoclonal antibody (anti‐HBs) directed against a single epitope on HBsAg in neutralizing the infectivity of Approximately 1,000 infectious doses of either HBV subtype adr or HBV subtype ayw.
Hepatitis B virus infects about 200 million people worldwide yearly. The consequences of the infection range from mild, self-limiting hepatitis with full recovery and immunity to chronic infection with liver disease of varying severity, including fulminant hepatitis and death. Alternatively, individuals may become healthy carriers of the virus and thus serve as a reservoir of infection. In addition, all chronic carriers of the virus are also at risk for development of primary hepatocellular carcinoma later in life. Efforts to combat this disease on a global scale require an inexpensive vaccine. Studies of the hepatitis B viral coat protein (HBsAg) have permitted the development of two generations of vaccine to date, and detailed structural studies of its antigenic sites may eventually allow the development of a completely synthetic (and therefore inexpensive) vaccine in the future.
Hepatitis B surface antigen possesses the group-specific determinant called a and one or another member from each of two pairs of allelic determinants, d and y as well as w and r, thereby creating the four major subtypes, adw, adr, ayw and ayr. In the sequence of major surface antigen polypeptides made of 226 amino acid residues, lysine or arginine at amino acid position 122 specifies d or y determinant, and lysine or arginine at position 160 specifies w or r determinant, respectively. By means of site-directed mutagenesis and expression of mutant genes in cultured cells, the mechanism for the loss of subtypic determinants on surface antigens was investigated at the molecular level. A rare sample of surface antigen of subtype ad, devoid of w or r determinant, had asparagine at position 160. When it was converted to lysine, the surface antigen of subtype adw was obtained. Two samples of surface antigen were subtyped as ar. They lacked d determinant, despite having lysine at position 122 which usually specified it. They differed from all reported sequences of surface antigen in amino acid 144 or 145. They displayed d determinant when amino acid 144 was converted from glutamic acid to aspartic acid, or when amino acid 145 was changed from alanine to glycine. These results indicate that the key amino acid residue at position 122 or 160 is indispensable for the expression of subtypic determinants and that some distant residues are also crucially involved in conforming them.
The molecular basis of the d or y immunological subtype of hepatitis B virus (HBV) surface antigen (HBsAg) has been investigated by mutation of specific amino acid residues. When combined with substitution of serine 113 by threonine, replacement of arginine 122 by lysine or of tyrosine 134 by phenylalanine, or both of these changes, altered the antigenic subtype of HBsAg from y+d- to y+d+. These same mutations had a more dramatic effect on the subtype of antibodies induced by the antigens, a combination of all three mutations completely changing the subtype from y to d. Our study thus identifies residues in HBsAg that not only affect the subtype but discriminate between changes in antigenic and immunogenic behaviour. It also shows how the y and d subtypes may be manifest by the same molecule.
DNA recombinant technology has radically changed hepatitis B virus (HBV) virology. The genetic organization, transcription and replication of the virus are basically understood, structures of integrated HBV sequences in hepatocellular carcinoma have been characterized, and new vaccines produced by recombinant DNA technique are being developed.
Closed-circular HBV DNA was introduced into cells of the established human hepatoma culture HepG2. The culture medium of one of 40 single-cell clones contained HBV surface antigen (HBsAg), core-related antigens (HBc/eAg), and HBV DNA sequences. HBV DNA and DNA polymerase activity were detected in particles resembling both nucleocapsids and complete virions (Dane particles). Intracellular integrated and extrachromosomal HBV DNA sequences were detected. Relaxed-circular and single-stranded forms of viral DNA were identified as likely replicative intermediates of the HBV genome. In conclusion, in vitro production of Dane-like particles by transformed human hepatocytes has been achieved. This model should be valuable as a cell culture system for studying virus replication and virus-host cell interactions.
Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also secreted from cells as a subviral
particle, without concomitant cleavage of N-terminal amino acid sequences. We examined this unusual export process in a cell-free
system and showed that the initial product of HBsAg biosynthesis is an integral transmembrane protein, with most or all of
its C-terminal half on the lumenal side of the endoplasmic reticulum membrane. To study the nature of its topogenic signals,
we synthesized fusion proteins between HBsAg and the nonsecreted protein alpha-globin. Fusion proteins in which approximately
100 amino acids of globin preceded all HBsAg sequences were successfully translocated in vitro; the same domain as in the
wild-type HBsAg was transported into the vesicle lumen. Fusions in which the entire globin domain was C terminal were able
to translocate both the C-terminal region of HBsAg and its attached globin domain. Thus, uncleaved signal sequences in p24s
function to direct portions of the molecule across the membrane and are able to perform this function even when positioned
in an internal protein domain.