Aptamer Binding Assay for the E Antigen of Hepatitis B Using Modified Aptamers with G-Quadruplex Structures.

The e antigen of hepatitis B (HBeAg) is positively associated with an increased risk of developing liver cancer and cirrhosis in chronic hepatitis B (CHB) patients. Clinical monitoring of HBeAg provides guidance to the treatment of CHB and the assessment of disease progression. We describe here an affinity binding assay for HBeAg, which takes advantage of G-quadruplex aptamers for enhanced binding and stability. We demonstrate a strategy to improve the binding affinity of aptamers by modifying their sequences upon their G-quadruplex and secondary structures. On the basis of predicting a stable G-quadruplex and a secondary structure, we truncated 19 nucleotides (nt) from the primer regions of an 80-nt aptamer, and the resulting 61-nt aptamer enhanced binding affinity by 19 times (Kd = 1.2 nM). We mutated a second aptamer (40 nt) in one loop region and incorporated pyrrolo-deoxycytidine to replace deoxycytidine in another loop. The modified 40-nt aptamer, with a stable G-quadruplex and two modified loops, exhibited a 100 times higher binding affinity for HBeAg (Kd = 0.4 nM) than the unmodified original aptamer. Using the two newly modified aptamers, one serving as the capture and the other as the reporter, we have developed an improved sandwich binding assay for HBeAg. Analyses of HBeAg in serum samples (concentration ranging from 0.1 to 60 ng/mL) of 10 hepatitis B patients, showing consistent results with clinical tests, demonstrate a successful application of the aptamer modification strategy and the associated aptamer binding assay.

Design, Synthesis and Bioactive Evaluation of Oxime Derivatives of Dehydrocholic Acid as Anti-Hepatitis B Virus Agents.

Oxime derivatives of dehydrocholic acid and its esters were designed for anti-hepatitis B virus (HBV) drugs according to principles of assembling active chemical fragments. Twelve compounds were synthesized from dehydrocholic acid by esterification and oxime formation, and their anti-hepatitis B virus (HBV) activities were evaluated with HepG 2.2.15 cells. Results showed that 5 compounds exhibited more effective inhibition of HBeAg than positive control, among them 2b-3 and 2b-1 showed significant anti-HBV activities on inhibiting secretion of HBeAg (IC50 (2b-3) = 49.39 ± 12.78 µM, SI (2b-3) = 11.03; IC50 (2b-1) = 96.64 ± 28.99 µM, SI (2b-1) = 10.35) compared to the Entecavir (IC50 = 161.24 µM, SI = 3.72). Molecular docking studies showed that most of these compounds interacted with protein residues of heparan sulfate proteoglycan (HSPG) in host hepatocyte and bile acid receptor.

Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy.

Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli, had unprecedentedly high binding affinities (Kd ∼10-12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

Anti-hepatitis B virus norbisabolane sesquiterpenoids from Phyllanthus acidus and the establishment of their absolute configurations using theoretical calculations.

Nineteen new highly oxygenated norbisabolane sesquiterpenoids, phyllanthacidoid acid methyl ester (1), and C-T (4-21), were isolated from Phyllanthus acidus Skeels, together with two known ones, phyllanthusols A (2) and B (3), whose sugar moiety was revised as glucosamine-N-acetate, rather than the previously assigned mannosamine-N-acetate. Compounds 2 and 3 were renamed respectively as phyllanthacidoids A (2) and B (3) to avoid confusion. All of the isolates except for 1 are glycosides, whose saccharide moieties possess a pentaoxy cyclohexane (scyllo quercitol) connecting with glucosamine-N-acetate or glucosyl moieties, which are first examples in natural products. Phyllanthacidoids N-R (15-19) with 8R configurations and/or 5,8-diketal skeleton, are unprecedented structures among norbisabolane sesquiterpenoids. Phyllanthacidoids S (20) and T (21) have the unusual tricyclo [3.1.1.1] oxygen bridge skeleton formed by a diketal system, of which the relative configurations of the aliphatic chain were assigned on the basis of heteronuclear coupling constants. The absolute configurations of compounds (1-21) were established by means of calculated electronic circular dichroism (ECD) and coupling constants. Compounds 1-5, 7-9, 10, and 14 displayed potential anti-hepatitis B virus (HBV) activities, with IC50 values of 0.8-36 µM against HBV surface antigen (HBsAg) and HBV excreted antigen (HBeAg), and the results indicated that the 5-ketal group and sugar moieties had contributions to the selectivity of HBsAg and HBeAg.

Hepatitis B virus e antigen physically associates with receptor-interacting serine/threonine protein kinase 2 and regulates IL-6 gene expression.

We previously reported that hepatitis B virus (HBV) e antigen (HBeAg) inhibits production of interleukin 6 by suppressing NF-κB activation. NF-κB is known to be activated through receptor-interacting serine/threonine protein kinase 2 (RIPK2), and we examined the mechanisms of interleukin 6 regulation by HBeAg. HBeAg inhibits RIPK2 expression and interacts with RIPK2, which may represent 2 mechanisms through which HBeAg blocks nucleotide-binding oligomerization domain-containing protein 1 ligand-induced NF-κB activation in HepG2 cells. Our findings identified novel molecular mechanisms whereby HBeAg modulates intracellular signaling pathways by targeting RIPK2, supporting the concept that HBeAg could impair both innate and adaptive immune responses to promote chronic HBV infection.

Human hepatitis B viral e antigen and its precursor P20 inhibit T lymphocyte proliferation.

The hepatitis B virus (HBV) Precore protein is processed through the secretory pathway directly as HBeAg or with the generation of an intermediate (P20). Precore gene has been shown to be implicated in viral persistence, but the functions of HBeAg and its precursors have not been fully elucidated. We show that the secreted proteins HBeAg and P20 interact with T cell surface and alter Kit-225 and primary T cells proliferation, a process which may facilitate the establishment of HBV persistence. Our data indicate that the N-terminal end of Precore is important for these inhibitory effects and exclude that they are dependent on the association of HBeAg and P20 with two characterized cell surface ligands, the Interleukin-1 Receptor Accessory Protein and gC1qR (present study).

Specific determination of hepatitis B e antigen by antibodies targeting precore unique epitope facilitates clinical diagnosis and drug evaluation against hepatitis B virus infection.

Hepatitis B e antigen (HBeAg) is a widely used marker both for chronic hepatitis B (CHB) clinical management and HBV-related basic research. However, due to its high amino acid sequence homology to hepatitis B core antigen (HBcAg), most of available anti-HBe antibodies are cross-reactive with HBcAg resulting in high interference against accurate measurement of the status and level of HBeAg. In the study, we generated several monoclonal antibodies (mAbs) targeting various epitopes on HBeAg and HBcAg. Among these mAbs, a novel mAb 16D9, which recognizes the SKLCLG (aa -10 to -5) motif on the N-terminal residues of HBeAg that is absent on HBcAg, exhibited excellent detection sensitivity and specificity in pairing with another 14A7 mAb targeting the HBeAg C-terminus (STLPETTVVRRRGR, aa141 to 154). Based on these two mAbs, we developed a novel chemiluminescent HBeAg immunoassay (NTR-HBeAg) which could detect HBeAg derived from various HBV genotypes. In contrast to widely used commercial assays, the NTR-HBeAg completely eliminated the cross-reactivity with secreted HBcAg from precore mutant (G1896A) virus in either cell culture or patient sera. The improved specificity of the NTR-HBeAg assay enables its applicability in cccDNA-targeting drug screening in cell culture systems and also provides an accurate tool for clinical HBeAg detection.

Bioinformatic analysis of the hepadnavirus e-antigen and its precursor identifies remarkable sequence conservation in all orthohepadnaviruses.

The hepatitis B e-antigen (HBeAg) is a non-particulate secretory protein expressed by all viruses within the family Hepadnaviridae. It is not essential for viral assembly or replication but is important for establishment of persistent infection in vivo. Although the exact mechanism(s) by which the HBeAg manifests chronicity are unclear, the HBeAg elicits both humoral and cell-mediated immunity, down-regulates the innate immune response to infection, as well as functioning as a T cell tolerogen and regulating the immune response to the intracellular nucleocapsid. A bioinformatics approach was used to show that the HBeAg and precursory genetic codes share remarkable sequence conservation in all mammalian-infecting hepadnaviruses, irrespective of host, genotype, or geographic origin. Whilst much of this sequence conservation was within key immunomodulatory epitopes, highest conservation was observed at the unique HBeAg N-terminus, suggesting this sequence in particular may play an important role in HBeAg function.

Capsids of hepatitis B virus e antigen with authentic C termini are stabilized by electrostatic interactions.

The hepatitis B virus e antigen, an alternative transcript of the core gene, is a secreted protein that maintains viral persistence. The physiological form has extended C termini relative to Cp(-10)149, the construct used in many studies. To examine the role of the C termini, we expressed the constructs Cp(-10)151 and Cp(-10)154, which have additional arginine residues. Both constructs when treated with reductant formed capsids more efficiently than Cp(-10)149. These capsids were also substantially more stable, as measured by thermal denaturation and resistance to urea dissociation. Mutagenesis suggests that electrostatic interactions between the additional arginine residues and glutamate residues on adjacent subunits play a role in the extra stabilization. These findings have implications for the physiological role and biotechnological potential of this protein.

Large-scale viral genome analysis identifies novel clinical associations between hepatitis B virus and chronically infected patients.

Despite the high global prevalence of chronic hepatitis B (CHB) infection, datasets covering the whole hepatitis B viral genome from large patient cohorts are lacking, greatly limiting our understanding of the viral genetic factors involved in this deadly disease. We performed deep sequencing of viral samples from patients chronically infected with HBV to investigate the association between viral genome variation and patients' clinical characteristics. We discovered novel viral variants strongly associated with viral load and HBeAg status. Patients with viral variants C1817T and A1838G had viral loads nearly three orders of magnitude lower than patients without those variants. These patients consequently experienced earlier viral suppression while on treatment. Furthermore, we identified novel variants that either independently or in combination with precore mutation G1896A were associated with the transition from HBeAg positive to the negative phase of infection. These observations are consistent with the hypothesis that mutation of the HBeAg open reading frame is an important factor driving CHB patient's HBeAg status. This analysis provides a detailed picture of HBV genetic variation in the largest patient cohort to date and highlights the diversity of plausible molecular mechanisms through which viral variation affects clinical phenotype.

Optimization of the expression of hepatitis B virus e gene in Pichia pastoris and immunological characterization of the product.

Escherichia coli-derived hepatitis B e antigen (HBeAg) is widely used for serological tests of hepatitis B virus (HBV). Because it exhibits cross-reactivity with HBcAb in human sera, current antibody to HBeAg (HBeAb) immunoassays are based on competitive inhibition enzyme-linked immunosorbent assay (ELISA) rather than sandwich ELISA, which interfere with the specificity and sensitivity of HBeAb detection. Pichia pastoris has advantages of eukaryotic cells, while having the capacity of high-level secretion of foreign proteins. To explore the diagnostic suitability of recombinant HBeAg (rHBeAg), we expressed the wild type HBV e gene (wt-e-gene) and the synthetic HBV e gene (syn-e-gene; native HBV e gene modified based on synonymous codon usage bias) in P. pastoris. The recombinant antigen was secreted into the medium. The expression level of rHBeAg was enhanced by optimizing HBV e gene. The yield of syn-e-gene product was approximately five-fold greater than wt-e-gene. The protein represented 66% of the total supernatant protein, and was simply purified to 90%. P. pastoris-derived HBeAg showed high HBe antigenicity, while lacking any HBc antigenicity and cross-reactivity between all proteins derived from the culture of P. pastoris and normal human sera. P. pastoris-derived HBeAg has higher specificity and sensitivity for detection HBeAb in the diagnostic assay than the commercial HBeAb ELISA kits.

Polyelectrolyte-protected Dual-color-quantum-dot Assembled Silica Nanoparticles and Their Application in Simultaneous Fluorescence Determination of e Antigen and Surface Antigen of Hepatitis B.

Cationic poly-diallyldimethylammonium (PDADMAC), green CdTe quantum dots (QDs) or red CdS coated CdTe QDs, and anionic polyacrylic acid (PAA) were respectively assembled on the nano-carrier SiO2 to prepare green fluorescence composite nanoparticles (GF-QDs) and red ones (RF-QDs) with the structure SiO2/PDADMAC/QD/PDADMAC/PAA. The sandwich structure "PDADMAC/QD/PDADMAC" on the nano-carrier not only realized the protection to fluorescence of QDs but also avoided the fluorescence shielding of silica shell for the assembled QDs. In 7 days, the diluent solutions of GF-QD and RF-QD all have a very stable fluorescence. On the contrary, the fluorescence of diluent solutions of red and green QDs reduced by 75.99 and 94.35%, respectively. Indeed, they have not fluorescent shielding and have a very slight fluorescent enhancement. Based on GF-QD and RF-QD, the simultaneous determination of Hepatitis B e antigen and surface antigen has been established. Their determination in buffer and plasma all showed good precision and accuracy.

Structures of Hepatitis B Virus Core- and e-Antigen Immune Complexes Suggest Multi-point Inhibition.

Hepatitis B virus (HBV) is the leading cause of liver disease worldwide. While an adequate vaccine is available, current treatment options are limited, not highly effective, and associated with adverse effects, encouraging the development of alternative therapeutics. The HBV core gene encodes two different proteins: core, which forms the viral nucleocapsid, and pre-core, which serves as an immune modulator with multiple points of action. The two proteins mostly have the same sequence, although they differ at their N and C termini and in their dimeric arrangements. Previously, we engineered two human-framework antibody fragments (Fab/scFv) with nano- to picomolar affinities for both proteins. Here, by means of X-ray crystallography, analytical ultracentrifugation, and electron microscopy, we demonstrate that the antibodies have non-overlapping epitopes and effectively block biologically important assemblies of both proteins. These properties, together with the anticipated high tolerability and long half-lives of the antibodies, make them promising therapeutics.

Ultrasensitive electrochemical immunosensor for quantitative detection of HBeAg using Au@Pd/MoS2@MWCNTs nanocomposite as enzyme-mimetic labels.

A sensitive sandwich-type electrochemical immunosensor for the detection of hepatitis B e antigen (HBeAg) was successfully developed based on the gold@palladium nanoparticles (Au@Pd NPs) loaded by molybdenum disulfide functionalized multiwalled carbon nanotubes (Au@Pd/MoS2@MWCNTs). The resultant nanocomposites not only possessed high specific surface area and good biocompatibility, but also exhibited excellent electro-catalytical property. Au NPs functionalized porous graphene oxide (p-GO@Au) were used as sensing platforms and primary antibodies carriers, which can accelerate the electron transfer and improve the load capacity of primary antibodies (Ab1), improving the sensitivity of the immunosensor. Under optimal conditions, the designed immunosensor could detect target HBeAg concentration in the range from 0.1pg/mL to 500pg/mL, with a low detection limit of 26fg/mL (S/N = 3) for HBeAg. Additionally, the designed immunosensor showed excellent specificity, good reproducibility and acceptable stability. The satisfactory results in analysis of human serum samples indicated that it had potential application in clinical monitoring of tumor markers.

Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy.

A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic 16O/18O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol-1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per µl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease.

Precore/core region mutations of hepatitis B virus related to clinical severity.

Despite the availability of an effective vaccine, hepatitis B virus (HBV) infection remains a major health problem, with more than 350 million chronically infected people worldwide and over 1 million annual deaths due to cirrhosis and liver cancer. HBV mutations are primarily generated due both to a lack of proofreading capacity by HBV polymerase and to host immune pressure, which is a very important factor for predicting disease progression and therapeutic outcomes. Several types of HBV precore/core (preC/C) mutations have been described to date. The host immune response against T cells drives mutation in the preC/C region. Specifically, preC/C mutations in the MHC class II restricted region are more common than in other regions and are significantly related to hepatocellular carcinoma. Certain mutations, including preC G1896A, are also significantly related to HBeAg-negative chronic infection. This review article mainly focuses on the HBV preC/C mutations that are related to disease severity and on the HBeAg serostatus of chronically infected patients.

Epitope mapping and serodiagnosis using Ata- and (Lys)7 peptides.

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.

Hepatitis B virus capsid: localization of the putative immunodominant loop (residues 78 to 83) on the capsid surface, and implications for the distinction between c and e-antigens.

Hepatitis B virus capsid protein comprises a 149 residue "assembly" domain that polymerizes into icosahedral particles, and a 34 residue RNA-binding "protamine" domain. Recently, the capsid structure has been studied to resolutions below 10 A by cryo-electron microscopy, revealing much of its alpha-helical substructure and that it appears to have a novel fold for a capsid protein; however, the resolution is still too low for chain-tracing by conventional criteria. Aiming to establish a fiducial marker to aid in the process of chain-tracing, we have used cryo-microscopy to pinpoint the binding site of a monoclonal antibody that recognizes the peptide from residues 78 to 83. This epitope resides on the outer rim of the 30 A long spikes that protrude from the capsid shell. These spikes are four-helix bundles formed by the pairing of helix-turn-helix motifs from two subunits; by means of a tilting experiment, we have determined that this bundle is right-handed. Variants of the same protein present two clinically important and non-crossreactive antigens: core antigen (HBcAg), which appears early in infection as assembled capsids; and the sentinel e-antigen (HBeAg), a non-particulate form. Knowledge of the binding site of our anti-HBcAg antibody bears on the molecular basis of the distinction between the two antigens, which appears to reflect conformational differences between the assembled and unassembled states of the capsid protein dimer, in addition to epitope masking in capsids.

Comparison of the Efficacy of Entecavir and Tenofovir in Nucleos(T)ide Analogue-Experienced Chronic Hepatitis B Patients.

The efficacy of entecavir (ETV) and tenofovir (TDF) for the treatment of nucleos(t)ide analogue (NA)-experienced chronic hepatitis B (CHB) patients has been little studied. Here, we compare the efficacy of both ETV and TDF in NA-experienced CHB patients without detectable genotypic resistance. This retrospective cohort study included consecutive NA-experienced patients who had neither current nor previous genotypic resistance and had received ETV or TDF for at least 6 months. Overall, 202 patients (146 patients in the ETV group and 56 in the TDF group) were analyzed. The cumulative probabilities of complete virologic suppression (CVS) at month 12 were 76.1% in the ETV group and 95.0% in the TDF group (P<0.001), respectively. The TDF-treated group achieved CVS more rapidly than the ETV group for both Hepatitis B e antigen (HBeAg)-negative and -positive patients (P = 0.006 and < 0.001, respectively), and for those with both low (< 2,000 IU/mL) and high (≥ 2,000 IU/mL) HBV DNA levels (P = 0.01 and 0.002, respectively). TDF group had an increased probability of achieving CVS (hazard ratio, 2.242; 95% confidence interval, 1.587-3.165; P = 0.001), after adjustment for HBV DNA level, the presence of HBeAg, and a history of CVS during prior treatment. During the treatment period, 23 patients (15.8%) in the ETV group developed virologic breakthrough, compared to none in the TDF group. The cumulative probabilities of developing virologic breakthrough and ETV-resistance at month 24 were 9.7% and 5.3%, respectively. In conclusion, TDF is preferable to ETV for achieving CVS in NA-experienced CHB patients without genotypic resistance.

Localization of the amino terminus of the hepatitis B virus core antigen within the core particle.

The position of the amino terminus of the hepatitis B virus core antigen (HBcAg) within the core particle was studied. For this purpose, three recombinant analogs of HBcAg were designed. One analog, HBcAgR, was identical in amino acid sequences to the core polypeptide of the hepatitis B virus; the second, HBeAgR, differed from the authentic protein in deletion of 39 carboxy-terminal amino acids. The amino acid sequences of the third polypeptide, HBe delta N and of HBeAgR were similar, HBe delta N differed from HBeAgR only in replacement of 3 N-terminal amino acids by 16 amino acids of beta-galactosidase. The HBcAg analogs were compared with respect to their reaction with monoclonal antibody (mAb E1A7) to the amino-terminal linear epitope of hepatitis B virus e antigen. Although able to assemble into virus-like particles, the three analogs of HBcAg, reacted differently with mAb E1A7. It was demonstrated that mAb E1A7 reacted with both native and denatured HBeAgR. HBe delta N was not recognized by mAb E1A7. In contrast, HBcAgR reacted with mAb E1A7 only when denatured. Native HBcAgR did not react with mAb E1A7 when assembled into particles. Thus evidence was obtained that the amino terminus of HBcAg is not exposed on the particle surface.