pre-S Region Research Articles

Male-Specific W4P/R Mutation in the Pre-S1 Region of Hepatitis B Virus, Increasing the Risk of Progression of Liver Diseases in Chronic Patients

The issue of hepatitis B virus (HBV) mutations possibly leading to a gender disparity in the progression of liver diseases has not been explored. We aimed to elucidate the relationships of the novel pre-S1 mutations, W4P/R, with the progression of liver diseases and male predominance in a South Korean chronic cohort by use of a molecular epidemiologic study. We developed a fluorescence resonance energy transfer (FRET)-based real-time PCR (RT-PCR) assay for the detection of the W4P/R mutations and applied it to 292 chronic HBV patients. The pre-S1 mutations from 247 (84.6%) of a total of 292 patients were detected by this assay. W4P/R mutants were found to be significantly related to severe liver diseases (hepatocellular carcinoma [HCC] and liver cirrhosis, 12.4% [19/153] of patients, versus chronic hepatitis and asymptomatic carriage, 1.1% [1/94] of patients) (P < 0.001). All of the W4P/R mutants were found in males only. The novel HBV pre-S1 mutations, W4P/R, may be associated with disease severity in male patients chronically infected with HBV genotype C. The W4P/R mutations may provide in part an explanation for the relatively high ratio of male to female incidence in HCC generation in South Korean chronic HBV patients.

Change in Hepatitis B Virus Large Surface Antigen Variant Prevalence 13 Years after Implementation of a Universal Vaccination Program in China

A nationwide hepatitis B virus (HBV) vaccination program was implemented in China starting in 1992. To study the change in HBV variant prevalence with massive immunization, large HBV surface protein (LHBs) genes from HBV surface antigen (HBsAg)-positive sera were amplified and sequenced. The prevalences of LHBs mutants were compared between the 1992 and 2005 surveys in child and adult groups. The prevalence of "α" determinant mutants in the children increased from 6.5% in 1992 to 14.8% in 2005, where the G145R mutant occurred most frequently. In contrast, mutation frequencies showed little difference between 1992 (9.4%) and 2005 (9.9%) in adults. Moreover, compared to the 1992 survey, the child group surface (S) protein mutation frequency specifically increased (P = 0.005) in the 2005 survey, but the pre-S region mutation frequency did not show a significant difference (P > 0.05). However, the mutation frequency in the adult group increased in both the pre-S and S regions. Furthermore, the frequencies of the disease-related pre-S2 deletion and start codon mutations were significantly higher in the adult groups than in the child groups in both the 1992 and 2005 surveys (P < 0.01). Massive immunization enhances the HBV S protein mutation; the prevalence of LHBs mutants, particularly disease-related mutants, tends to increase with patient age.

P36: Occupational blood exposure and compliance to universal precautions: a cross‐sectional survey among healthcare workers in Beijing, China

PURPOSE OF THE STUDY: The goals of our study were to evaluate HBV genotypes and recombinants; mutations related to antiviral resistance, hepatic disease progression and vaccine escape in HBV monoinfected patients in S~ ao Paulo city, Brazil. METHODS: Full-length or partial HBV sequences from 39 treated and untreated patients were performed (1). Mutations were mapped and phylogenetic analyses performed. RESULTS: Twenty-eight full-length and 11 partial HBV sequences (HBsAg and the RT domain) were obtained. The genotype distribution was: A (51.3%), D (25.6%), F (15.4%) and C (7.7%). Primary resistance mutations were not found, however, we found compensatory mutations in 43% of the treated patients and 12.5% among untreated patients. We also found mutations related to changes in the HBsAg antigenicity (vaccine escape and decrease of HBsAg detection in serological tests); disease progression in the precore/core gene (85.7%), including A1762T/ G1764A/G1896A mutations in HBsAg positive and negative patients; and deletions in the Pre-S1 and Pre-S2 region (14%), more specifically in the start codons and the S gene promoter. CONCLUSIONS: The genotype distribution (A, D, F and C) reflects the historical context of the population from S~ ao Paulo, which is composed of immigrants from Africa (A), Europe (D) and Asia (C), and migrants, mostly coming from the Northeast (A) and North of Brazil (F), being the Amerindian population common in the North region. Most of the S gene mutations, which lead to the HBsAg antigenicity changes, are the characteristics that distinguish the different genotypes, which might explain the antigenicity differences among them. A1762T/G1764A/G1896A mutations in the core/precore region are not common in HBeAg positive patients, and G1896A mutation is not common in genotypes A and F either, but it was found in this study. In the analysis of the nucleotide 1858 in the two sequences with G1896A mutation, genotypes A and F respectively, T1858 was found instead of C1858, which would allow the selection of the G1896A mutation in these genotypes. The Pre-S deletions in start codons and A1762T/G1764A/G1896A mutations in positive HBeAg patients may be related to non-majoritary viral populations and need further studies using next-generation sequencing technologies. REFERENCE

The hepatitis B virus preS1 domain hijacks host trafficking proteins by motif mimicry

Hepatitis B virus (HBV) is an infectious, potentially lethal human pathogen. However, there are no effective therapies for chronic HBV infections. Antiviral development is hampered by the lack of high-resolution structures for essential HBV protein-protein interactions. The interaction between preS1, an HBV surface-protein domain, and its human binding partner, γ2-adaptin, subverts the membrane-trafficking apparatus to mediate virion export. This interaction is a putative drug target. We report here atomic-resolution descriptions of the binding thermodynamics and structural biology of the interaction between preS1 and the EAR domain of γ2-adaptin. NMR, protein engineering, X-ray crystallography and MS showed that preS1 contains multiple γ2-EAR-binding motifs that mimic the membrane-trafficking motifs (and binding modes) of host proteins. These motifs localize together to a relatively rigid, functionally important region of preS1, an intrinsically disordered protein. The preS1-γ2-EAR interaction was relatively weak and efficiently outcompeted by a synthetic peptide. Our data provide the structural road map for developing peptidomimetic antivirals targeting the γ2-EAR-preS1 interaction.

A complete genomic analysis of hepatitis B virus isolated from 516 Chinese patients with different clinical manifestations

This study investigated features and clinical implications of HBV mutations in patients with different clinical manifestations. In total, 516 patients were enrolled in this study, including 131 patients with acute hepatitis B, 239 patients with chronic hepatitis B, and 146 patients with acute-on-chronic liver failure. HBV genotypes and mutations were analyzed by direct sequencing of complete viral genomes. Genotypes B2, C1, C2, and D1 accounted for 22.2%, 1.6%, 74.6%, and 1.6%, respectively. Genotype B was more frequently detected in patients with acute hepatitis B than those with chronic hepatitis B and acute-on-chronic liver failure. Deletion mutations were detected mostly in preS1 and preS2 regions and the detection rates were 3.8%, 19.7%, and 24.7% for acute hepatitis B, chronic hepatitis B and acute-on-chronic liver failure patients, respectively. Incidences of point mutation T53C (preS1F53L), G1613A (polR841K), G1775A and A1762T + G1764A in the basal core promoter region, G1896A and G1899A in precore region and A2189C (coreI97L) in core region increased along with acute hepatitis B, chronic hepatitis B, and acute-on-chronic liver failure. The mutation G1896A was independently associated with poor survival of patients with acute-on-chronic liver failure. The gradual increase of viral mutation incidences was also observed in three HLA-A2-restricted cytotoxic T lymphocyte epitopes from HLA-A2-positive patients, that is env188-196 (5.8%, 10.1%, 22.5%), core107-115 (4.3%, 4.6%, 19.7%), and x92-100 (1.4%, 20.2%, 33.8%). In conclusion, certain viral mutations in various regions of HBV genome are associated with disease progression of HBV infection.

HBV Subgenotypes D1, D2, D-del! Are ‘Old‘ Genotyping Methods Interpreted Correctly?

HBV Subgenotypes D1, D2, D-del! Are ‘Old‘ Genotyping Methods Interpreted Correctly?

A monoclonal antibody specific to the non-epitope region of hepatitis B virus preS1 contributes to more effective HBV detection

ObjectivesThe hepatitis B virus (HBV) preS1 protein is divided into an epitope region and a non-epitope region based on the respective antigenicities of these regions. Most of the antibodies that are currently used to detect the large surface protein of HBV (HBV LHB) are specific to the epitope region of preS1, which may contribute to the false negative results of HBV LHB detection assays. Here, we established a mouse monoclonal antibody (mAb) that could improve the efficiency of HBV LHB detection. Design and methodsThe HBV preS1 protein was expressed in E. coli strain BL21 and used to screen hybridoma clones. HBV preS1-specific mAb was produced by immunizing mice with a chemically synthesized peptide antigen derived from the non-epitope region of HBV preS1. The mAb was characterized by ELISA, Western blot, and immunocytochemistry and was subsequently used in serum sample tests. ResultsBased on in silico B cell epitope predictions, the HBV preS1 aa 91–117 peptide was synthesized as an antigen. Recombinant HBV preS1 was expressed in E. coli and identified by SDS-PAGE. The mAb D8 (IgG2b) recognized the recombinant preS1 protein in both ELISA and Western blot assays and also recognized the preS1 protein expressed in plasmid-transfected HepG2.2.15 cells by immunocytochemistry. Furthermore, the D8 mAb, which is specific for the non-epitope region of preS1, contributed to the improved sensitivity and specificity of HBV detection. ConclusionsWe established an mAb that is specific to the non-epitope region of HBV preS1 and improved the detection of HBV LHB in an ELISA assay. This mAb could help increase the accuracy of the clinical measurement of preS1.

Combined core promoter mutations and pre‐S deletion of HBV may not increase the risk of HCC: a geographical epidemiological study in Guangxi, China

Although persistent hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), the mechanisms of oncogenesis remain obscure. To determine whether the findings that HBV basal core promoter (BCP) A1762T, G1764A double mutations, pre-S deletions and a combination of both are risk factors of HCC are supported by geographical epidemiology. Study subjects were recruited from Long An county, where the incidence of HCC is the highest, and five other counties in Guangxi, where the HCC incidence is lower and varies among them. The Pre-S region and BCP of HBV from all study subjects were amplified and sequenced and the data were analysed using chi-squared tests. The prevalence of BCP and pre-S mutations differs significantly (χ(2) = 9.850, 5.135, respectively, all P < 0.01) between Long An and the other counties. However, the prevalence of combined BCP and pre-S mutations does not differ significantly (χ(2) = 1.510, P > 0.05). These mutations are less frequent in the young but the prevalence of pre-S deletions does not increase with age. The prevalence of these mutations does not differ significantly between men and women but is significantly higher in Zhuang than the other ethnic populations. Among the other five counties, the prevalence of BCP mutations in counties where the HCC incidence is high is significantly higher than that of counties where the HCC incidence is low. Combined BCP double mutations and pre-S deletion may not increase the risk of HCC, although these mutations are a risk factor of HCC when they present alone.

A bile acid transporter as a candidate receptor for hepatitis B and D virus entry

Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.

Naturally Occurring Hepatitis B Virus B‐Cell and T‐Cell Epitope Mutants in Hepatitis B Vaccinated Children

To control hepatitis B virus (HBV) infection, a universal HBV vaccination program for infants was launched in Taiwan in 1984. The aim of this study was to investigate the role of B-cell and T-cell epitope variations of HBsAg and polymerase in HBV infection in vaccinated children. One hundred sixty-three sera from vaccinated children were enrolled randomly. HBV serum markers, including hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) and core antigen (anti-HBc), were detected by ELISA. Nucleotide sequences encoding the S and the pre-S regions of HBsAg were analyzed in all HBsAg positive sera. Five children were HBsAg positive. Sequence analysis of S, pre-S, and overlapped polymerase (P) genes showed that HBV isolates of HBsAg-positive vaccinees were variants; no G145R but G145A and other substitutions were found in the “a” determinant. Fifteen, six, and eight amino acid substitutions within B-cell and T-cell epitopes of S, pre-S, and P regions were detected, respectively. Several immune-epitope mutants, such as S45T/A, N131T, I194V, and S207N in S, were detected in all isolates. In conclusion, our results suggested that these naturally occurring immunoepitope mutants, which changed their immunogenicity leading to escape from immune response, might cause HBV infection.

Whole genome HBV deletion profiles and the accumulation of preS deletion mutant during antiviral treatment

BackgroundHepatitis B virus (HBV), because of its error-prone viral polymerase, has a high mutation rate leading to widespread substitutions, deletions, and insertions in the HBV genome. Deletions may significantly change viral biological features complicating the progression of liver diseases. However, the clinical conditions correlating to the accumulation of deleted mutants remain unclear. In this study, we explored HBV deletion patterns and their association with disease status and antiviral treatment by performing whole genome sequencing on samples from 51 hepatitis B patients and by monitoring changes in deletion variants during treatment. Clone sequencing was used to analyze preS regions in another cohort of 52 patients.ResultsAmong the core, preS, and basic core promoter (BCP) deletion hotspots, we identified preS to have the highest frequency and the most complex deletion pattern using whole genome sequencing. Further clone sequencing analysis on preS identified 70 deletions which were classified into 4 types, the most common being preS2. Also, in contrast to the core and BCP regions, most preS deletions were in-frame. Most deletions interrupted viral surface epitopes, and are possibly involved in evading immuno-surveillance. Among various clinical factors examined, logistic regression showed that antiviral medication affected the accumulation of deletion mutants (OR = 6.81, 95% CI = 1.296 ~ 35.817, P = 0.023). In chronic carriers of the virus, and individuals with chronic hepatitis, the deletion rate was significantly higher in the antiviral treatment group (Fisher exact test, P = 0.007). Particularly, preS2 deletions were associated with the usage of nucleos(t)ide analog therapy (Fisher exact test, P = 0.023). Dynamic increases in preS1 or preS2 deletions were also observed in quasispecies from samples taken from patients before and after three months of ADV therapy. In vitro experiments demonstrated that preS2 deletions alone were not responsible for antiviral resistance, implying the coordination between wild type and mutant strains during viral survival and disease development.ConclusionsWe present the HBV deletion distribution patterns and preS deletion substructures in viral genomes that are prevalent in northern China. The accumulation of preS deletion mutants during nucleos(t)ide analog therapy may be due to viral escape from host immuno-surveillance.

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.

Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.

Effects of Genomic Changes in Hepatitis B Virus on Postoperative Recurrence and Survival in Patients with Hepatocellular Carcinoma

To determine whether the genomic changes in hepatitis B virus (HBV) affect the clinical outcomes of hepatocellular carcinoma (HCC) in patients with HBV-associated HCC treated with curative surgical resection. A total of 247 patients with HBV-associated HCC were treated with curative surgical resection. They were followed regularly for a median of 30months. The whole X, S, basal core promoter (BCP), and precore regions of HBV were sequenced. The genomic changes such as the G1896A at precore, the A1762T/G1764A at BCP, the C1653T and the T1753V at X gene, and pre-S2 deletion were not significantly associated with postoperative recurrence of HCC or survival of patients after curative resection. However, in univariate analysis, younger age, elevated serum α-fetoprotein level, elevated serum alanine aminotransferase level, larger tumor size, microvascular invasion, and advanced Cancer of the Liver Italian Program stage were closely associated with shorter survival after surgical resection. In multivariate analysis, only microvascular invasion revealed to be an independent risk factor of postoperative recurrence (relative risk [RR] 5.406; P<0.001); the independent risk factors of shorter survival appeared to be infiltrative type (RR 5.110; P=0.032), larger tumor size (RR 1.976; P=0.047), and microvascular invasion (RR 6.118; P<0.001). The postoperative recurrence or survival period may not be affected by the genomic changes at the precore, BCP, X, and pre-S2 regions in HBV of genotype C2 in patients with HBV-associated HCC treated with curative surgical resection. Rather, it may be closely associated with tumor characteristics, such as the size and type of HCC or presence of microvascular invasion.

Spectroscopic Characterization and Fusogenic Properties of PreS Domains of Duck Hepatitis B Virus

In order to shed light on the hepatitis B virus fusion mechanism and to explore the fusogenic capabilities of preS regions, a recombinant duck hepatitis B virus (DHBV) preS protein (DpreS) containing six histidines at the carboxy-terminal end has been obtained. The DpreS domain, which has an open and mostly nonordered conformation as indicated by fluorescence and circular dichroism spectroscopies, has the ability to interact with negatively charged phospholipid vesicles. The observed interaction differences between neutral and acidic phospholipids can be interpreted in terms of an initial ionic interaction between the phospholipid polar headgroup and the protein followed by the insertion of probably the N-terminal region in the cellular membrane. Fluorescence polarization studies detect a decrease of the transition enthalpy together with a small modification of the transition temperature, typical effects of integral membrane proteins. The interaction of the protein with acidic phospholipid vesicles induces aggregation, lipid mixing, and leakage of internal contents, properties that have been ascribed to membrane destabilizing proteins. The fact that the preS domains of the hepadnaviruses have little similarity but share a very similar hydrophobic profile points to the importance of the overall three-dimensional structure as well as to its conformational flexibility and the distribution of polar and apolar amino acids on the expression of their destabilizing properties rather than to a particular amino acid sequence. The results presented herein argue for the involvement of DpreS in the initial steps of DHBV infection. Taken together with previously reported results, the conclusion that both S and preS regions participate in the fusion process of the hepadnaviridae family may be drawn.

Tracking the naturally occurring mutations across the full-length genome of hepatitis B virus of genotype D in different phases of chronic e-antigen-negative infection

Hepatitis B e-antigen (HBeAg)-negative chronic HBV infection is highly prevalent in several parts of the world, including India, with the clinical spectrum ranging from inactive carrier (IC) state to chronic ‘e-negative’ hepatitis B (CHB) and culminating in advanced liver disease such as cirrhosis (LC). The present study has for the first time investigated the natural diversity of HBV belonging to genotype D in treatment-naïve Indian patients representing the above phases of HBeAg-negative infection to identify candidate mutations associated with each disease state. Studies of full-length HBV/D sequences revealed that the progressive accumulation and persistence of mutations in basal core promoter, negative regulatory element, Pre-core region, the B- and T-cell epitopes of X protein as well as deletions in the PreS region contribute significantly to disease progression from IC through CHB to LC. In addition, the development of CHB was associated with a significant increase in viral variants characterized by mutations in enhancer II, preS1 promoter, T-cell epitope of core and B-cell epitope region of PreS1. While few of the mutations were previously reported in the context of HBV genotypes B and C, others had not been documented before. Our results thus highlight a distinct pattern of mutation in HBV/D that may help in predicting clinical outcomes of HBeAg-negative infection and have implications for better clinical management of the patients.

Removing N-Terminal Sequences in Pre-S1 Domain Enhanced Antibody and B-Cell Responses by an HBV Large Surface Antigen DNA Vaccine

Although the use of recombinant hepatitis B virus surface (HBsAg) protein vaccine has successfully reduced global hepatitis B infection, there are still a number of vaccine recipients who do not develop detectable antibody responses. Various novel vaccination approaches, including DNA vaccines, have been used to further improve the coverage of vaccine protection. Our previous studies demonstrated that HBsAg-based DNA vaccines could induce both humoral and CMI responses in experimental animal models. However, one form of the the HBsAg antigen, the large S antigen (HBs-L), expressed by DNA vaccine, was not sufficiently immunogenic in eliciting antibody responses. In the current study, we produced a modified large S antigen DNA vaccine, HBs-L(T), which has a truncated N-terminal sequence in the pre-S1 region. Compared to the original HBs-L DNA vaccine, the HBs-L(T) DNA vaccine improved secretion in cultured mammalian cells and generated significantly enhanced HBsAg-specific antibody and B cell responses. Furthermore, this improved HBsL DNA vaccine, along with other HBsAg-expressing DNA vaccines, was able to maintain predominantly Th1 type antibody responses while recombinant HBsAg protein vaccines produced in either yeast or CHO cells elicited mostly Th2 type antibody responses. Our data indicate that HBsAg DNA vaccines with improved immunogenicity offer a useful alternative choice to recombinant protein-based HBV vaccines, particularly for therapeutic purposes against chronic hepatitis infection where immune tolerance led to poor antibody responses to S antigens.

Impact of hepatitis B virus (HBV) preS/S genomic variability on HBV surface antigen and HBV DNA serum levels

To evaluate whether hepatitis B virus (HBV) preS/S gene variability has any impact on serum hepatitis B surface antigen (HBsAg) levels and to analyze the replication capacity of naturally occurring preS/S variants, sera from 40 untreated patients with HBV-related chronic liver disease (hepatitis B e antigen [HBeAg]-positive, n = 11; HBeAg-negative, n = 29) were virologically characterized. Additionally, phenotypic analysis of three different preS/S variant isolates (carrying a 183-nucleotide deletion within the preS1 region, the deletion of preS2 start codon, and a stop signal at codon 182 within the S gene, respectively) was performed. HBV infecting 14 (35%) patients had single or multiple preS/S genomic mutations (i.e., preS1 and/or preS2 deletions, preS2 start codon mutations, C-terminally truncated and/or "a" determinant mutated S protein). Presence of preS/S variants negatively correlated with HBsAg titers (r = -0.431; P = 0.005) and its prevalence did not significantly differ between HBeAg-positive and HBeAg-negative patients. No correlation was found between HBsAg and HBV DNA levels in patients infected with preS/S mutants, whereas a significant correlation was found between HBsAg and viremia levels (r = 0.607; P = 0.001) in patients infected with wild-type HBV strains. HepG2 cells replicating the above-mentioned three preS/S variants showed significant reduction of HBsAg secretion, retention of envelope proteins in the endoplasmic reticulum, less efficient virion secretion and nuclear accumulation of significantly higher amounts of covalently closed circular DNA compared with wild-type HBV replicating cells. In patients infected with preS/S variants, HBV DNA replication and HBsAg synthesis/secretion appear to be dissociated. Therefore, the use of HBsAg titer as diagnostic/prognostic tool has to take into account the frequent emergence of preS/S variants in chronic HBV infection.

Genetic diversity of hepatitis B virus genotypes B6, D and F among circumpolar indigenous individuals

Hepatitis B virus (HBV) infection is highly prevalent in circumpolar indigenous peoples. However, the clinical outcome is extremely variable, such that while hepatocellular carcinoma (HCC) is uncommon in Canadian Inuit, the incidence of HCC is slightly higher in Greenlanders than in Danes, and it is especially high in Alaskan Native people infected with HBV genotypes F (HBV/F) and C (HBV/C). These differences may be associated with the genomic variability of the predominant HBV genotype in each group. The purpose of this study was to determine the rate, nature and regional susceptibility of HBV genomic mutations among circumpolar indigenous individuals. Paired serum samples, separated by 5-6 years, were analysed from Canadian and Greenlandic Inuit infected with HBV genotype B6 (HBV/B6) and HBV/D, respectively, and from Alaskan Native people infected with HBV/F, each having subsequently developed HCC. Phylogenetic and mutational analyses were performed on full-genome sequences, and the dynamic evolution within the quasispecies population of each patient group was determined by clonal analysis of the non-overlapping core coding region. Mutations associated with severe outcomes predominated in HBV/F, mostly within the precore/core and PreS1 region. HBV/B6 genomes exhibited higher diversity compared to HBV/D and HBV/F, particularly within the core coding region. Thus, differing mutational profiles and genetic variability were observed among different HBV genotypes predominating in circumpolar indigenous patients. The unusual observation of persistently high genetic variability with HBV/B6 despite clinical inactivity could be due to the evolution of a host-pathogen balance, but other possible factors also need to be explored.

Structures of Hepatitis B Virus Cores Presenting a Model Epitope and Their Complexes with Antibodies

The core shell of hepatitis B virus is a potent immune stimulator, giving a strong neutralizing immune response to foreign epitopes inserted at the immunodominant region, located at the tips of spikes on the exterior of the shell. Here, we analyze structures of core shells with a model epitope inserted at two alternative positions in the immunodominant region. Recombinantly expressed core protein assembles into T = 3 and T = 4 icosahedral shells, and atomic coordinates are available for the T = 4 shell. Since the modified protein assembles predominantly into T = 3 shells, a quasi-atomic model of the native T = 3 shell was made. The spikes in this T = 3 structure resemble those in T = 4 shells crystallized from expressed protein. However, the spikes in the modified shells exhibit an altered conformation, similar to the DNA containing shells in virions. Both constructs allow full access of antibodies to the foreign epitope, DPAFR from the preS1 region of hepatitis B virus surface antigen. However, one induces a 10-fold weaker immune response when injected into mice. In this construct, the epitope is less constrained by the flanking linker regions and is positioned so that the symmetry of the shell causes pairs of epitopes to come close enough to interfere with one another. In the other construct, the epitope mimics the native epitope conformation and position. The interaction of native core shells with an antibody specific to the immunodominant epitope is compared to the constructs with an antibody against the foreign epitope. Our findings have implications for the design of vaccines based on virus-like particles.

Combination and evolution of HBV mutant strains in the HBeAg-positive status predict clinical outcomes after HBeAg seroconversion.

We investigated whether the combined presence and evolution of hepatitis B virus (HBV) mutant strains in the hepatitis B e antigen (HBeAg)-positive status can predict clinical outcomes after HBeAg seroconversion. One hundred and eighty-six patients with spontaneous HBeAg seroconversion were enrolled into this longitudinal study. The sequences of pre-S, core promoter, and precore regions were determined at study entry and at the visit immediately before HBeAg seroconversion. Age ≥40years at HBeAg seroconversion, male sex, and higher HBV DNA levels at entry were independent predictors for HBeAg-negative chronic hepatitis B (CHB). Patients with combined mutations of pre-S deletions and T1762/A1764 had a significantly increased risk of cirrhosis and hepatocellular carcinoma (HCC) compared to patients with the wild type at both genomic regions. Combinations of pre-S deletions and T1762/A1764 were found on the same HBV genome by cloning analysis of full-length HBV genomes. Patients with a persistent presence of pre-S deletions and T1762/A1764 mutations, and new development of pre-S deletions in the HBeAg-positive status were significantly at an increased risk of HBeAg-negative CHB, cirrhosis, and HCC after HBeAg seroconversion than those with a persistent presence of the wild type at both genomic regions. After adjusting the other risk factors, the evolution of pre-S deletions was an independent predictor for cirrhosis [hazard ratio (HR): 1.52, 95% confidence interval (CI) 1.02-2.25] and HCC (HR: 4.0, 95% CI 1.6-10.1). The combined presence and evolution of pre-S deletions and T1762/A1764 in the HBeAg-positive status was a useful factor significantly predictive of clinical outcomes in patients with spontaneous HBeAg seroconversion.