HBV Pre-S1-Derived Myristoylated Peptide (Myr47): Identification of the Inhibitory Activity on the Cellular Uptake of Lipid Nanoparticles.

The Myr47 lipopeptide, consisting of hepatitis B virus (HBV) pre-S1 domain (myristoylated 2-48 peptide), is an effective commercialized anti-HBV drug that prevents the interaction of HBV with sodium taurocholate cotransporting polypeptide (NTCP) on human hepatocytes, an activity which requires both N-myristoylation residue and specific amino acid sequences. We recently reported that Myr47 reduces the cellular uptake of HBV surface antigen (HBsAg, subviral particle of HBV) in the absence of NTCP expression. In this study, we analyzed how Myr47 reduces the cellular uptake of lipid nanoparticles (including liposomes (LPs) and HBsAg) without NTCP expression. By using Myr47 mutants lacking the HBV infection inhibitory activity, they could reduce the cellular uptake of LPs in an N-myristoylation-dependent manner and an amino acid sequence-independent manner, not only in human liver-derived cells but also in human non-liver-derived cells. Moreover, Myr47 and its mutants could reduce the interaction of LPs with apolipoprotein E3 (ApoE3) in an N-myristoylation-dependent manner regardless of their amino acid sequences. From these results, lipopeptides are generally anchored by inserting their myristoyl residue into the lipid bilayer and can inhibit the interaction of LPs/HBsAg with apolipoprotein, thereby reducing the cellular uptake of LPs/HBsAg. Similarly, Myr47 would interact with HBV, inhibiting the uptake of HBV into human hepatic cells, while the inhibitory effect of Myr47 may be secondary to its ability to protect against HBV infection.

An integrated vitamin E-coated polymer hybrid nanoplatform: A lucrative option for an enhanced in vitro macrophage retention for an anti-hepatitis B therapeutic prospect.

A platform capable of specifically delivering an antiviral drug to the liver infected with hepatitis B is a major concern in hepatology. Vaccination has had a major effect on decreasing the emerging numbers of new cases of infection. However, the total elimination of the hepatitis B virus from the body requires prolonged therapy. In this work, we aimed to target the liver macrophages with lipid polymer hybrid nanoparticles (LPH), combining the merit of polymeric nanoparticles and lipid vesicles. The hydrophilic antiviral drug, entecavir (E), loaded LPH nanoparticles were optimized and physicochemically characterized. A modulated lipidic corona, as well as, an additional coat with vitamin E were used to extend the drug release enhance the macrophage uptake. The selected vitamin E coated LPH nanoparticles enriched with lecithin-glyceryl monostearate lipid shell exhibited high entrapment for E (80.47%), a size ≤ 200 nm for liver passive targeting, extended release over one week, proven serum stability, retained stability after refrigeration storage for 6 months. Upon macrophage uptake in vitro assessment, the presented formulation displayed promising traits, enhancing the cellular retention in J774 macrophages cells. In vivo and antiviral activity futuristic studies would help in the potential application of the ELPH in hepatitis B control.

Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.

The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.

A hepatitis B virus-derived human hepatic cell-specific heparin-binding peptide: identification and application to a drug delivery system.

Viruses are naturally evolved nanocarriers that can evade host immune systems, attach specifically to the surfaces of target cells, enter the cells through endocytosis, escape from endosomes efficiently, and then transfer their genomes to host cells. Hepatitis B virus (HBV) is a ∼42 nm enveloped DNA virus that can specifically infect human hepatic cells. To utilize the HBV-derived early infection machinery in synthetic nanocarriers, the human hepatic cell-binding site (i.e., the sodium taurocholate co-transporting polypeptide (NTCP)-binding site, with myristoylated pre-S1(2-47)) and the low pH-dependent fusogenic domain (pre-S1(9-24)) are indispensable for targeting and endosomal escape, respectively. However, cell-surface NTCP has recently been shown not to be involved in the initial attachment of HBV. In this study, we identified a novel heparin-binding site (pre-S1(30-42)) in the N-terminal half of the pre-S1 region, which presumably interacts with cell-surface heparan sulfate proteoglycan (HSPG) and plays a pivotal role in the initial attachment of HBV to human hepatic cells. The evolutionarily conserved amino acid residues Asp-31, Trp-32, and Asp-33 are indispensable for the heparin-binding activity. Liposomes (LPs) displaying the peptide were endocytosed by human hepatic cells in a cell-surface heparin-dependent manner and delivered doxorubicin to human hepatic cells more efficiently than myristoylated pre-S1(2-47)-displaying LPs. These results demonstrated that the pre-S1(30-42) peptide is the most promising HBV-derived targeting peptide for synthetic nanocarriers, and that this peptide exhibits high specificity for human hepatic cells and efficiently induces endocytosis.

Analysis of gingival crevicular fluid as applied to the diagnosis of oral and systemic diseases.

Gingival crevicular fluid (GCF), a serum transudate or inflammatory exudate, can be collected from the gingival crevice surrounding the teeth. As such, the fluid reflects the constituents of serum, the cellular response in the periodontium, and contributions from the gingival crevice. The study of GCF has focused on defining the pathophysiology of periodontal disease, and identification of a potential diagnostic test for active periodontitis. The majority of markers that have been identified as potential candidates for such a test are measures of inflammation (i.e., prostaglandin E2 (PGE2), neutrophil elastase, and the lysosomal enzyme beta-glucuronidase). Further, analysis of inflammatory markers in GCF may assist in defining how certain systemic disorders (e.g., diabetes mellitus) can modify periodontal disease, and how periodontal disease/periodontal inflammation can influence certain systemic disorders (i.e., cardiovascular/cerebrovascular diseases). Methodological concerns related to the collection and analysis of GCF are important factors that need to be considered when studying GCF. Practical concerns argue against the widespread clinical application of GCF as an adjunct to periodontal diagnosis. Rather, analysis of GCF-derived mediators in saliva may serve as a means of rapid screening for periodontal disease.

Heparin at physiological concentration can enhance PEG-free in vitro infection with human hepatitis B virus.

Hepatitis B virus (HBV) is a blood-borne pathogen responsible for chronic hepatitis, cirrhosis, and liver cancer. The mechanism of HBV entry into hepatocytes remains to be investigated. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was discovered as a major HBV receptor based on an in vitro infection system using NTCP-reconstituted HepG2 cells. However, this infection system relies on the compound polyethylene glycol (4% PEG), which is not physiologically relevant to human infection. High concentration of heparin has been commonly used as an inhibitor control for in vitro infection in the field. Surprisingly, we found that heparin at physiological concentration can enhance HBV infection in a PreS1-peptide sensitive, NTCP-dependent manner in both HepaRG and HepG2-NTCP-AS cells. O-sulfation of heparin is more important for the infection enhancement than N-sulfation. This system based on the HepG2-NTCP-AS cells can support in vitro infection with HBV genotypes B and C, as well as using serum samples from HBeAg positive and negative chronic carriers. In summary, our study provides a PEG-free infection system closely resembling human natural infection. In addition, it points to a future research direction for heparin and heparin-binding host factor(s) in the blood, which are potentially involved in viral entry. To our knowledge, this is the first soluble and circulatory host factor which can enhance HBV in vitro infection.

[Management of hepatitis B virus and hepatitis C virus infection in chronic kidney failure]. / Prise en charge de l'infection par les virus des hépatites B ou C chez l'insuffisant rénal chronique.

Chronic infections by hepatitis B (HBV) and C virus (HCV) result in diagnosis and therapeutic issues in dialysis and kidney recipients patients. The exposure to nosocomial, including blood transfusion, risk explains the high prevalence of HBV and HCV infection in this setting. Chronic infection reduces the survival of both patients and allografts, including a specific risk of de novo glomerulonephritis. Cirrhosis was considered as a contra-indication to renal transplantation given the high risk of decompensation and death, questionning the indication of a combined liver and kidney transplantation. Thus, it is mandatory to screen HBV and HCV markers in all dialysis patients, whether or not they are candidates to transplantation. Liver biopsy allows evaluating the severity of the liver disease since the noninvasive markers of fibrosis appear to be less accurate in "renal" patients than in the general population and to better define antiviral therapeutic indications. HCV treatment was mainly based on pegylated interferon α (and low doses of ribavirin), which is contra-indicated in kidney recipients given the risk of graft rejection; HCV treatment is now based on the use of oral direct acting antivirals, which are very potent and well tolerated. HBV replication is now easily suppressed by second-generation nucleos(t)tidic analogues (entecavir and tenofovir), which will be indicated in all the dialysis patients with significant fibrosis (F2,3 or 4 according to the Metavir scoring system) and in any candidate to renal transplantation and to any HBsAg-positive kidney recipients. The best treatment remains preventive by anti-HBV vaccination for HBV and by the respect of universal hygiene rules for HCV.

Antiviral activity of phosphatidyl-dideoxycytidine in hepatitis B-infected cells and enhanced hepatic uptake in mice.

Dideoxycytidine (ddC) inhibits the replication of hepatitis B virus (HBV) but its clinical use is limited by peripheral neuropathy. We synthesized dioleoylphosphatidyl-ddC (DOP-ddC), a phospholipid prodrug of ddC which forms lipid bilayers and is readily incorporated into liposomes. The 90% effective dose (ED90) of DOP-ddC was 18 microM vs. 7 microM for ddC. However, in HBV-infected human hepatoma cells (2.2.15 cells), DOP-ddC was less toxic in vitro. When liposomal DOP-[5,6-3H]ddC was administered intraperitoneally to mice, drug levels in liver were 40 times greater than [5,6-3H]ddC when expressed as area under curve. Liposomal DOP-ddC also provided higher levels of drug in lymph nodes and spleen, important accessory sites of HBV replication. Plasma levels of drug remained above the ED90 six times longer with DOP-ddC than with ddC. DOP-ddC levels in sciatic nerve, the major site of toxicity, were not significantly different from levels observed with free ddC. The phospholipid prodrug approach is a general one which may readily be applied to other antiviral nucleosides for HBV.

Effects of subchronic treatment with natural human interferons on antipyrine clearance and liver function in patients with chronic hepatitis.

To determine whether natural human interferon administered under the usual therapeutic dosing scheme would inhibit the hepatic drug metabolism, we performed an antipyrine test in eight patients with chronic B or non-A, non-B hepatitis before and after a subchronic interferon therapy (6 megaunits/day for 17 +/- 4 days, mean +/- SD). Six patients received interferon-beta and 2 received interferon-alpha. To circumvent a possible influence of interferon-induced fever on the hepatic drug metabolism, the antipyrine test during the interferon therapy was performed at least 14 days after the interferon-induced fever disappeared. The kinetic parameters of antipyrine were obtained from seven saliva samples over 32 hours postdose. There were no significant differences in any kinetic parameters of antipyrine observed before and during the interferon therapy. With the sample size of the study, there was only a 20% chance (i.e., beta-power = 0.8 at alpha = 0.05) that we might have missed a 17% reduction in antipyrine clearance by the interferon therapy (type II error). On the other hand, the subchronic interferon therapy lowered serum aminotransferases and DNA polymerase activity significantly (P less than .05) compared with the respective baseline values. Our results suggest that the subchronic therapeutic dosing scheme of interferon as conducted in the present study does not cause the inhibitory effect on the oxidative drug metabolism to a statistically significant or clinically relevant degree in patients with chronic hepatitis, while it improves their liver function. Further studies are required for determining if different types of interferons administered under the different dosing schemes would alter the hepatic drug metabolism and the inhibitory effect would be time-dependent.

Comparative salivary proteome of hepatitis B- and C-infected patients.

Hepatitis B and C virus (HBV and HCV) infections are an important cause of cirrhosis and hepatocellular carcinoma. The natural history has a prominent latent phase, and infected patients may remain undiagnosed; this situation may lead to the continuing spread of these infections in the community. Compelling reasons exist for using saliva as a diagnostic fluid because it meets the demands of being an inexpensive, noninvasive and easy-to-use diagnostic method. Indeed, comparative analysis of the salivary proteome using mass spectrometry is a promising new strategy for identifying biomarkers. Our goal is to apply an Orbitrap-based quantitative approach to explore the salivary proteome profile in HBV- and HCV-infected patients. In the present study, whole saliva was obtained from 20 healthy, (control) 20 HBV-infected and 20 HCV-infected subjects. Two distinct pools containing saliva from 10 subjects of each group were obtained. The samples were ultracentrifuged and fractionated, and all fractions were hydrolyzed (trypsin) and injected into an LTQ-VELOS ORBITRAP. The identification and analyses of peptides were performed using Proteome Discoverer1.3 and ScaffoldQ + v.3.3.1. From a total of 362 distinct proteins identified, 344 proteins were identified in the HBV, 326 in the HCV and 303 in the control groups. Some blood proteins, such as flavin reductase (which converts biliverdin to bilirubin), were detected only in the HCV group. The data showed a reduced presence of complement C3, ceruloplasmin, alpha(1)-acid glycoprotein and alpha(2)-acid glycoprotein in the hepatitis-infected patients. Peptides of serotransferrin and haptoglobin were less detected in the HCV group. This study provides an integrated perspective of the salivary proteome, which should be further explored in future studies targeting specific disease markers for HBV and HCV infection.

Study of detergent-mediated liberation of hepatitis B virus-like particles from S. cerevisiae homogenate: identifying a framework for the design of future-generation lipoprotein vaccine processes.

Virus-like particles (VLPs) are expressed intracellularly in Saccharomyces cerevisiae and the recovery process involves the use of a detergent, which facilitates the release of VLP from host cell components. The detergent-mediated liberation of VLPs is a critical step in primary recovery and is responsible for setting the backdrop for subsequent purification in terms of product yield and characteristics of the process stream. In this paper the use of Triton X-100 detergent for the recovery of lipid envelope VLPs, using the hepatitis B surface antigen (HBsAg) as the VLP model, was investigated. To develop a framework that can be adopted in process design for future generation VLP vaccine candidates, the impact of Triton X-100 was characterized via different response factors: (i) recovery and activity of the HBsAg; (ii) level of protein and lipid contamination from the host cell; and (iii) indirect impact on the performance of an ultrafiltration step following primary recovery. Our studies identified that an increase in detergent concentration favors recovery of HBsAg only to a specific threshold, 0.5% v/v Triton X-100. Further increase in detergent results in delipidation of HBsAg leading to loss in antigenic activity. The level of contamination due to host protein and lipid co-liberation is in proportion with the amount of detergent employed. Greater membrane resistance during ultrafiltration was observed for samples generated using higher concentrations of detergent due to the increase in membrane fouling by the contaminants. Based on this study, Triton X-100 concentrations in the range of 0.2-0.5% v/v appears to be most suitable for recovery of native HBsAg. Choosing between 0.2-0.5% v/v would involve identifying a suitable tradeoff between desired product yield and the level of contamination that can be tolerated by downstream operations.

Interaction between desialylated hepatitis B virus and asialoglycoprotein receptor on hepatocytes may be indispensable for viral binding and entry.

The cellular receptor for hepatitis B virus (HBV) infection has not yet been identified. The purpose of this study was to address the possibility of participation by desialylated HBV and the asialoglycoprotein receptor (ASGP-R) exclusively expressed on liver parenchymal cells, in infection. Assays for viral binding and entry were performed by culturing a hepatoblastoma cell line, HepG2, and HBV particles derived from the HBV carrier in the presence or absence of neuraminidase (NA). Viral binding and entry were clearly enhanced in the presence of NA, and the enhancement of the binding could be blocked by asialo-fetuin and ethylenediamine-tetraacetic acid (EDTA). In addition, covalently closed circular (CCC)-DNA, as a marker of infectivity, was detected in the presence of NA, but not in its absence. The optimal concentration of NA raised infectivity more than 1000 times. We concluded that this method makes it feasible to evaluate the infectivity of HBV in vitro and that ASGP-R may be a specific HBV receptor once viral particles are desialylated.

Significance of natural polymerized albumin and its receptor in hepatitis B infection of hepatocytes.

Lack of information regarding the presence of native albumin polymer in serum and its structural similarity to the one produced by glutaraldehyde treatment casts doubt on the postulate that hepatitis B virus attachment to hepatocytes is mediated through polymerized albumin. We used a sandwich enzyme-linked immunosorbent assay with murine monoclonal antibodies raised against glutaraldehyde-polymerized albumin to detect native albumin polymer in human serum and its cross-reactivity with other albumin polymers. Presence of polymerized albumin receptor on the HepG2 cell was studied by radioreceptor assay. Purified hepatitis B virus and synthetic peptide analogous to part of pre-S2 sequence (120-145) were used to study polymerized albumin-dependent attachment of the virus to HepG2 cells. Antibodies raised against pre-S2 peptide were used to inhibit the pre-S2 and hepatitis B virus attachment to HepG2 cells. Glutaraldehyde-treated polymerized albumin was found to be immunologically cross-reactive with native albumin polymer. Its levels were found to be significantly raised in sera of patients with liver diseases. Polymerized albumin has specific saturable receptor on HepG2 cells with two classes of binding sites of different equilibrium dissociation constant (Kd1 = (16 +/- 9.6)pmol/L and Kd2 = (1,019 +/- 172)pmol/L. Albumin monomer was unable to compete for the polymerized albumin receptor sites on HepG2 cells. Anti-pre-S2 antibodies inhibit hepatitis B virus and pre-S2 binding to hepatocyte by 40% and 70%, respectively. Added extraneous polymerized albumin and the antibody against it did not interfere with virus attachment to HepG2 cells.