Biomedical subjects

Erik Depla

Publications and source records attributed to Erik Depla.

12 recordsLinked to original sources

PubMed2006

Viral and cellular determinants of the hepatitis C virus envelope-heparan sulfate interaction.

Cellular binding and entry of hepatitis C virus (HCV) are the first steps of viral infection and represent a major target for antiviral antibodies and novel therapeutic strategies. We have recently demonstrated that heparan sulfate (HS) plays a key role in the binding of HCV envelope glycoprotein E2 to target cells (Barth et al., J. Biol. Chem. 278:41003-41012, 2003). In this study, we characterized the HCV-HS interaction and analyzed its inhibition by antiviral host immune responses. Using recombinant envelope glycoproteins, virus-like particles, and HCV pseudoparticles as model systems for the early steps of viral infection, we mapped viral and cellular determinants of HCV-HS interaction. HCV-HS binding required a specific HS structure that included N-sulfo groups and a minimum of 10 to 14 saccharide subunits. HCV envelope binding to HS was mediated by four viral epitopes overlapping the E2 hypervariable region 1 and E2-CD81 binding domains. In functional studies using HCV pseudoparticles, we demonstrate that HCV binding and entry are specifically inhibited by highly sulfated HS. Finally, HCV-HS binding was markedly inhibited by antiviral antibodies derived from HCV-infected individuals. In conclusion, our results demonstrate that binding of the viral envelope to a specific HS configuration represents an important step for the initiation of viral infection and is a target of antiviral host immune responses in vivo. Mapping of viral and cellular determinants of HCV-HS interaction sets the stage for the development of novel HS-based antiviral strategies targeting viral attachment and entry.

Animals↗

PubMed2006

Host cell responses induced by hepatitis C virus binding.

Initiation of hepatitis C virus (HCV) infection is mediated by docking of the viral envelope to the hepatocyte cell surface membrane followed by entry of the virus into the host cell. Aiming to elucidate the impact of this interaction on host cell biology, we performed a genomic analysis of the host cell response following binding of HCV to cell surface proteins. As ligands for HCV-host cell surface interaction, we used recombinant envelope glycoproteins and HCV-like particles (HCV-LPs) recently shown to bind or enter hepatocytes and human hepatoma cells. Gene expression profiling of HepG2 hepatoma cells following binding of E1/E2, HCV-LPs, and liver tissue samples from HCV-infected individuals was performed using a 7.5-kd human cDNA microarray. Cellular binding of HCV-LPs to hepatoma cells resulted in differential expression of 565 out of 7,419 host cell genes. Examination of transcriptional changes revealed a broad and complex transcriptional program induced by ligand binding to target cells. Expression of several genes important for innate immune responses and lipid metabolism was significantly modulated by ligand-cell surface interaction. To assess the functional relevance and biological significance of these findings for viral infection in vivo, transcriptional changes were compared with gene expression profiles in liver tissue samples from HCV-infected patients or controls. Side-by-side analysis revealed that the expression of 27 genes was similarly altered following HCV-LP binding in hepatoma cells and viral infection in vivo. In conclusion, HCV binding results in a cascade of intracellular signals modulating target gene expression and contributing to host cell responses in vivo. Reprogramming of cellular gene expression induced by HCV-cell surface interaction may be part of the viral strategy to condition viral entry and replication and escape from innate host cell responses.

Antigens, Viral↗

PubMed2005

Humoral immune response in acute hepatitis C virus infection.

BACKGROUND: There is little information on the timing, magnitude, specificity, and clinical relevance of the antibody response to acute hepatitis C virus (HCV) infection. We investigated the specificity, titer, and neutralizing potential of antibody responses to acute infection by examining 12 injection drug users before, during, and after infection. METHODS: Seroconversion was defined as incident detection of HCV-specific antibodies by using a commercially available enzyme-linked immuosorbent assay (ELISA). HCV protein-specific antibody responses were measured using recombinant antigens in an ELISA. For neutralization assays, plasma was incubated with human immunodeficiency virus (HIV)-HCV H77 or control HIV-murine leukemia virus (MLV) pseudotype virus and then allowed to infect Hep3B hepatoma cells. RESULTS: The mean time to HCV seroconversion was 6 weeks after the onset of viremia. Antibody responses to nonstructural proteins were detected before responses to the structural proteins, and antibodies to both were primarily restricted to the immunoglobulin G1 (IgG1) subclass. The maximum median end point titers for antibody responses to structural and nonstructural proteins were 1 : 600 and 1 : 6400, respectively. Antibodies that neutralized a retroviral pseudotype bearing HCV 1a envelope glycoproteins were detected at seroconversion in only 1 subject and at 6-8 months after seroconversion in 3 subjects. The delayed appearance of neutralizing antibodies was consistent with the late development of antibodies specific for the viral envelope glycoproteins, which are believed to mediate virus neutralization. CONCLUSION: The humoral immune response to acute HCV infection is of relatively low titer, is restricted primarily to the IgG1 subclass, and is delayed. A better understanding of why production of neutralizing antibody is delayed may improve efforts to prevent HCV infection.

Acute Disease↗

PubMed2005

Uptake and presentation of hepatitis C virus-like particles by human dendritic cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Interaction of dendritic cells (DCs) with viral particles may play an important role in the immunopathogenesis of HCV infection. Since the synthesis or purification of infectious virions is limited, we used HCV-like particles (HCV-LPs) to study the interaction of HCV with human DCs. Immature DCs exhibited an envelope-specific and saturable binding of HCV-LPs, indicating receptor-mediated DC-HCV-LP interaction. Confocal microscopy revealed that HCV-LPs were rapidly taken up by DCs in a temperature-dependent manner. Competition experiments demonstrated that C-type lectins such as mannose receptor or DC-SIGN (DC-specific intercellular adhesion molecule 3-grabbing nonintegrin) were not sufficient for mediating HCV-LP binding. HCV-LP uptake was followed by DC activation. DCs pulsed with HCV-LPs stimulated HCV core-specific CD4(+) T cells, indicating that uptake of HCV-LPs by DCs leads to antigen processing and presentation on major histocompatibility complex (MHC) class II molecules. Finally, HCV-LP-derived antigens were efficiently cross-presented to HCV core-specific CD8(+) T cells. These findings demonstrate that HCV-LPs represent a novel model system to study HCV-DC interaction allowing definition of the molecular mechanisms of HCV uptake, DC activation, and antigen presentation to T cells. Furthermore, HCV-LP-mediated DC activation and efficient antigen presentation may explain the marked immunogenicity of HCV-LPs in vivo.

Antigen Presentation↗

PubMed2004

A candidate vaccine based on the hepatitis C E1 protein: tolerability and immunogenicity in healthy volunteers.

The tolerability and immunogenicity of the hepatitis C virus E1 protein as a candidate vaccine was examined in a Phase I, single-arm study. Twenty healthy male volunteers were injected in the deltoid muscle at weeks 0, 3 and 6 with 20 microg recombinant E1 adsorbed on alum. A fourth (booster) dose was administered to 19 subjects at week 26. The candidate therapeutic vaccine was well tolerated. Three vaccine doses induced a clear humoral anti-E1 response that was boosted by a fourth dose. A strong, specific cellular immune response towards E1 was elicited in all vaccine recipients, which included a clear Th1 type response in all but one of the subjects.

Adolescent↗

PubMed2004

Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation.

Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to "hide" within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection.

Cell Adhesion Molecules↗

PubMed2004

Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C.

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies.

Acute Disease↗

PubMed2003

Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.

The conservation of positively charged residues in the N terminus of the hepatitis C virus (HCV) envelope glycoprotein E2 suggests an interaction of the viral envelope with cell surface glycosaminoglycans. Using recombinant envelope glycoprotein E2 and virus-like particles as ligands for cellular binding, we demonstrate that cell surface heparan sulfate proteoglycans (HSPG) play an important role in mediating HCV envelope-target cell interaction. Heparin and liver-derived highly sulfated heparan sulfate but not other soluble glycosaminoglycans inhibited cellular binding and entry of virus-like particles in a dose-dependent manner. Degradation of cell surface heparan sulfate by pretreatment with heparinases resulted in a marked reduction of viral envelope protein binding. Surface plasmon resonance analysis demonstrated a high affinity interaction (KD 5.2 x 10-9 m) of E2 with heparin, a structural homologue of highly sulfated heparan sulfate. Deletion of E2 hypervariable region-1 reduced E2-heparin interaction suggesting that positively charged residues in the N-terminal E2 region play an important role in mediating E2-HSPG binding. In conclusion, our results demonstrate for the first time that cellular binding of HCV envelope requires E2-HSPG interaction. Docking of E2 to cellular HSPG may be the initial step in the interaction between HCV and the cell surface resulting in receptor-mediated entry and initiation of infection.

Cell Line↗

PubMed2003

A pilot study of therapeutic vaccination with envelope protein E1 in 35 patients with chronic hepatitis C.

New treatments are needed for chronic hepatitis C patients in whom viral clearance cannot be achieved. Thirty-five chronic hepatitis C patients (genotype 1) were randomized to receive 20 mug of recombinant HCV E1 (E1) (n = 26) or placebo (n = 9) intramuscularly at weeks 0, 4, 8, 12, and 24. Thirty-four then received open-label E1 vaccine at weeks 50, 53, 56, 59, 62, and 65. Twenty-four patients (12 men, 12 women; mean age, 52 y; 18 interferon-based treatment failures; mean baseline alanine aminotransferase [ALT] level, 118 IU/L) underwent a biopsy before and after 2 courses of E1, 17 months later. Liver histology was scored by 2 blinded pathologists according to the Ishak and Metavir systems. Postinjection reactions were similar to placebo (alum only). Nine of 24 patients (38%) had improvement of 2 points or more, 10 (41%) remained stable, and 5 (21%) showed worsening in total Ishak score. Nine patients (38%) improved both on Ishak and Metavir fibrosis scores. Plasma HCV-RNA levels remained unchanged, whereas ALT levels showed a trend toward a decrease during treatment. All but 3 patients developed a significant de novo E1-specific T-cell response. The increase in anti-E1 antibody levels correlated with the decrease in total Ishak score and with the relative decreases in both Ishak fibrosis score and ALT level (all P < or =.01). In conclusion, E1 therapeutic vaccination is well tolerated and the observed effects warrant further study.

Alanine Transaminase↗

PubMed2002

Ultrastructural visualization of hepatitis C virus components in human and primate liver biopsies.

BACKGROUND/AIMS: Molecular and structural studies of hepatitis C virus (HCV) replication and infection have been performed on cultured cells and on serum of infected patients. No conclusive studies were conducted yet on human liver biopsies. This paper describes the ultrastructural findings of hepatitis C virus components in liver biopsies. METHODS: Liver specimens from acutely and chronically HCV-infected chimpanzees (five each) and 29 chronic hepatitis C patients were studied. Diagnosis of HCV infection was based on clinical, serological, light microscopic and immunohistochemical data and on HCV RNA polymerase chain reaction. RESULTS: In HCV-infected chimpanzees, tubular aggregates were observed in the cytoplasm of a significant number of hepatocytes and proven by immuno-electron microscopy to contain HCV-E2 viral envelope material. Identical tubular aggregates were seen in hepatocytes of chronic HCV-infected patients, although in smaller quantity and less frequently. A few single enveloped virus-like particles of 50-60 nm in diameter were seen for the first time in the hyaloplasm of hepatocytes of HCV-infected chimpanzees and patients. CONCLUSIONS: For the first time, HCV envelope material was ultrastructurally identified in hepatocytes of HCV-infected chimpanzees and patients. Virus-like particles, although strongly suggestive for HCV, failed final confirmation at least by routinely used methods.

Animals↗

PubMed2002

Detection of neutralizing antibodies to hepatitis C virus using a biliary cell infection model.

The identification and characterization of neutralizing anti-hepatitis C virus (HCV) antibodies may have a major impact on understanding HCV pathogenesis. However, to date, their detection has only been based on the inhibition of either the E2 envelope protein or HCV virions binding to different target cells. The permissiveness of primary biliary cells for HCV infection has been demonstrated previously. In the present report, infection of biliary cells was demonstrated further by combining PCR and immunohistochemical detection of the HCV core protein. This study demonstrates, using both serum and purified IgG, the presence of neutralizing anti-HCV antibodies in the serum of patients showing long-term response to antiviral therapy. Overall, the usefulness of the primary biliary cell infection model to investigate anti-HCV neutralization is shown.

Adult↗

PubMed2002

Binding of hepatitis C virus-like particles derived from infectious clone H77C to defined human cell lines.

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of viral entry and infection has been hampered by the inability to efficiently propagate the virus in cultured cells and the lack of a small-animal model. Recent studies have shown that in insect cells, the HCV structural proteins assemble into HCV-like particles (HCV-LPs) with morphological, biophysical, and antigenic properties similar to those of putative virions isolated from HCV-infected humans. In this study, we used HCV-LPs derived from infectious clone H77C as a tool to examine virus-cell interactions. The binding of partially purified particles to human cell lines was analyzed by fluorescence-activated cell sorting with defined monoclonal antibodies to envelope glycoprotein E2. HCV-LPs demonstrated dose-dependent and saturable binding to defined human lymphoma and hepatoma cell lines but not to mouse cell lines. Binding could be inhibited by monoclonal anti-E2 antibodies, indicating that the HCV-LP-cell interaction was mediated by envelope glycoprotein E2. Binding appeared to be CD81 independent and did not correlate with low-density lipoprotein receptor expression. Heat denaturation of HCV-LPs drastically reduced binding, indicating that the interaction of HCV-LPs with target cells was dependent on the proper conformation of the particles. In conclusion, our data demonstrate that insect cell-derived HCV-LPs bind specifically to defined human cell lines. Since the envelope proteins of HCV-LPs are presumably presented in a virion-like conformation, the binding of HCV-LPs to target cells may allow the study of virus-host cell interactions, including the isolation of HCV receptor candidates and antibody-mediated neutralization of binding.

Animals↗