glycoprotein-e2--hepatitis-c-virus and Hepatitis-C

Hepatitis C virus (HCV) is a major global health concern, and though therapeutic options have improved, no vaccine is available despite decades of research. As HCV can rapidly mutate to evade the immune response, an effective HCV vaccine must rely on identification and characterization of sites critical for broad immune protection and viral neutralization. This knowledge depends on structural and mechanistic insights of the E1 and E2 envelope glycoproteins, which assemble as a heterodimer on the surface of the virion, engage coreceptors during host cell entry, and are the primary targets of antibodies. Due to the challenges in determining experimental structures, structural information on E1 and E2 and their interaction is relatively limited, providing opportunities to model the structures, interactions, and dynamics of these proteins. This review highlights efforts to model the E2 glycoprotein structure, the assembly of the functional E1E2 heterodimer, the structure and binding of human coreceptors, and recognition by key neutralizing antibodies. We also discuss a comparison of recently described models of full E1E2 heterodimer structures, a simulation of the dynamics of key epitope sites, and modeling glycosylation. These modeling efforts provide useful mechanistic hypotheses for further experimental studies of HCV envelope assembly, recognition, and viral fitness, and underscore the benefit of combining experimental and computational modeling approaches to reveal new insights. Additionally, computational design approaches have produced promising candidates for epitope-based vaccine immunogens that specifically target key epitopes, providing a possible avenue to optimize HCV vaccines versus using native glycoproteins. Advancing knowledge of HCV envelope structure and immune recognition is highly applicable toward the development of an effective vaccine for HCV and can provide lessons and insights relevant to modeling and characterizing other viruses.

Topics: Binding Sites; Hepacivirus; Hepatitis Antibodies; Hepatitis C; Humans; Models, Molecular; Protein Binding; Protein Conformation; Protein Multimerization; Quantitative Structure-Activity Relationship; Structure-Activity Relationship; Viral Envelope Proteins

The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are critical in viral attachment and cell fusion, and studies of these proteins may provide valuable insights into their potential uses in vaccines and antiviral strategies. Progress has included elucidating the crystal structures of portions of their ectodomains, as well as many other studies of hypervariable regions, stem regions, glycosylation sites, and the participation of E1/E2 in viral fusion with the endosomal membrane. The available structural data have shed light on the binding sites of cross-neutralizing antibodies. A large amount of information has been discovered concerning heterodimerization, including the roles of transmembrane domains, disulfide bonding, and heptad repeat regions. The possible organization of higher order oligomers within the HCV virion has also been evaluated on the basis of experimental data. In this review, E1/E2 structure and function is discussed, and some important issues requiring further study are highlighted.

Topics: Animals; Antiviral Agents; Hepacivirus; Hepatitis C; Humans; Protein Domains; Viral Envelope Proteins; Viral Vaccines

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. HCV is able to evade host defense mechanisms, including both innate and acquired immune responses, to establish persistent infection, which results in a broad spectrum of pathogenicity, such as lipid and glucose metabolism disorders and hepatocellular carcinoma development. The HCV genome is characterized by a high degree of genetic diversity, which can be associated with viral sensitivity or resistance (reflected by different virological responses) to interferon (IFN)-based therapy. In this regard, it is of importance to note that polymorphisms in certain HCV genomic regions have shown a close correlation with treatment outcome. In particular, among the HCV proteins, the core and nonstructural proteins (NS) 5A have been extensively studied for their correlation with responses to IFN-based treatment. This review aims to cover updated information on the impact of major HCV genetic factors, including HCV genotype, mutations in amino acids 70 and 91 of the core protein and sequence heterogeneity in the IFN sensitivity-determining region and IFN/ribavirin resistance-determining region of NS5A, on virological responses to IFN-based therapy.

Topics: Animals; Antiviral Agents; Drug Resistance, Viral; Genotype; Hepacivirus; Hepatitis C; Humans; Interferons; Mutation; Phenotype; Treatment Outcome; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins

Hepatitis C virus (HCV), a member of the family Flaviviridae, is a leading cause of chronic liver disease and cancer. Recent advances in HCV therapeutics have resulted in improved cure rates, but an HCV vaccine is not available and is urgently needed to control the global pandemic. Vaccine development has been hampered by the lack of high-resolution structural information for the two HCV envelope glycoproteins, E1 and E2. Recently, Kong and coworkers (Science 342:1090-1094, 2013, doi:10.1126/science.1243876) and Khan and coworkers (Nature 509[7500]:381-384, 2014, doi:10.1038/nature13117) independently determined the structure of the HCV E2 ectodomain core with some unexpected and informative results. The HCV E2 ectodomain core features a globular architecture with antiparallel β-sheets forming a central β sandwich. The residues comprising the epitopes of several neutralizing and nonneutralizing human monoclonal antibodies were also determined, which is an essential step toward obtaining a fine map of the human humoral response to HCV. Also clarified were the regions of E2 that directly bind CD81, an important HCV cellular receptor. While it has been widely assumed that HCV E2 is a class II viral fusion protein (VFP), the newly determined structure suggests that the HCV E2 ectodomain shares structural and functional similarities only with domain III of class II VFPs. The new structural determinations suggest that the HCV glycoproteins use a different mechanism than that used by class II fusion proteins for cell fusion.

Topics: Animals; Hepacivirus; Hepatitis C; Humans; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Virus; Viral Envelope Proteins

For a long time, the lack of an appropriate cell culture system has hampered the study of neutralizing antibody responses against hepatitis C virus (HCV). However, the last decade has seen the development of several model systems that have significantly advanced our understanding of viral entry and antibody neutralization. Studies of acutely infected patients suggest that a strong and early production of neutralizing antibodies may contribute to control the virus during the acute phase of HCV infection and facilitate viral elimination by cellular immune responses. It also emerges that the early antibody response mainly targets hypervariable region 1 (HVR1) of the envelope glycoprotein E2. This host response can lead to viral escape from neutralization by rapid amino acid changes in this hypervariable region. In contrast, cross-reactive neutralizing antibodies seem to appear later during HCV infection, and several mechanisms contribute to reduce their accessibility to their cognate epitopes. These include the masking of major conserved neutralizing epitopes by HVR1, specific N-linked glycans and the lipid moiety of the viral particle. Other potential mechanisms of evasion from the neutralizing antibody response include a modulation by high-density lipoproteins and interfering antibodies as well as the capacity of the virus to be transferred by cell-to-cell contacts. Finally, the recent identification of several highly conserved neutralizing epitopes provides some opportunities for the design and development of vaccine candidates that elicit a protective humoral immune response.

Topics: Antibodies, Neutralizing; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatocytes; Humans; Immune Evasion; Tetraspanin 28; Viral Envelope Proteins; Viral Proteins; Virus Internalization

Hepatitis C virus (HCV) is the major cause of chronic liver disease as well as the major indication for liver transplantation worldwide. Current standard of care is not completely effective, not administrable in grafted patients, and burdened by several side effects. This incomplete effectiveness is mainly due to the high propensity of the virus to continually mutate under the selective pressure exerted by the host immune response as well as currently administered antiviral drugs. The E2 envelope surface glycoprotein of HCV (HCV/E2) is the main target of the host humoral immune response and for this reason one of the major variable viral proteins. However, broadly cross-neutralizing monoclonal antibodies (mAbs) directed against HCV/E2 represent a promising tool for the study of virus-host interplay as well as for the development of effective prophylactic and therapeutic approaches. In the last few years many anti-HCV/E2 mAbs have been evaluated in preclinical and clinical trials as possible candidate antivirals, particularly for administration in pre- and post-transplant settings. In this review we summarize the antigenic and structural characteristics of HCV/E2 determined through the use of anti-HCV/E2 mAbs, which, given the absence of a crystal structure of this glycoprotein, represent currently the best tool available.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Epitopes; Hepacivirus; Hepatitis C; Humans; Viral Envelope Proteins; Viral Proteins

Hepatitis C virus, a major human pathogen, produces infectious virus particles with several unique features, such as an ability to interact with serum lipoproteins, a dizzyingly complicated process of virus entry, and a pathway of virus assembly and release that is closely linked to lipoprotein secretion. Here, we review these unique features, with an emphasis on recent discoveries concerning virus particle structure, virus entry and virus particle assembly and release.

Topics: Animals; Endosomes; Hepacivirus; Hepatitis C; Humans; Mice; Models, Biological; Receptors, Virus; Tetraspanin 28; Viral Envelope Proteins; Virus Assembly; Virus Internalization; Virus Release

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease and the most common indication for liver transplantation. Current therapies are ineffective in a relevant percentage of patients raising the urgent medical need to develop adequate therapies for this infection. Broadly neutralizing human monoclonal antibodies (mAbs) directed against the HCV E2 glycoprotein (HCV/E2), the major target of the neutralizing humoral immune response, are considered as a possible novel therapeutic strategy for this infection. In the last few years, several anti-HCV/E2 human mAbs have been described in literature to be possibly used for therapeutic or prophylactic purposes. In this review, we illustrate the best candidates for an anti-HCV mAb-based therapy, considering their cross-neutralization profiles and their ability to overcome possible viral escape mechanisms.

Topics: Animals; Antibodies, Monoclonal; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Viral Envelope Proteins

CD81, a member of the tetraspanin integral membrane protein family, has been identified as an essential receptor for HCV (hepatitis C virus). The present review highlights recent published data on the role that CD81 plays in HCV entry, including the importance of actin-dependent lateral diffusion of CD81 within the cell membrane, CD81 endocytosis and the CD81-Claudin-1 receptor complex in HCV internalization. Additional functions for CD81 in the viral life cycle and the role of HCV-CD81 interactions in HCV-induced B-cell and CNS (central nervous system) abnormalities are discussed.

Topics: Animals; Antigens, CD; Hepacivirus; Hepatitis C; Humans; Liver; Protein Binding; Tetraspanin 28; Viral Envelope Proteins; Virus Internalization

The entry of hepatitis C virus (HCV) into host cells is an obligatory step in virus replication that presents a multi-faceted opportunity for drug discovery. However, the current understanding of HCV entry is rudimentary at best, with insights to date obtained by examining the fusion of pseudoparticles that express the HCV surface glycoproteins E1 and E2. The absence of an infectious virus replication system capable of replicating through a full virus life-cycle has hampered progress in determining the events involved in viral entry, resulting in considerable ambiguity surrounding the process. The recent development of an infectious HCV virus replication system provides a method with which to examine HCV entry in detail in a setting that promises greater authenticity and with the potential to increase understanding of this process. Weak inhibitors of the interactions between the HCV glycoproteins and potential host cell receptors have been identified, but their mechanism of action, in the context of virus infectivity, is not understood, and these inhibitors remain to be validated with infectious virus in cell culture. The advent of an infectious virus replication system holds considerable promise for developing a detailed understanding of HCV entry, but while mechanistic insights developed with other viruses may provide useful paradigms for experimental design, it is likely that HCV entry will be characterized by several unique biochemical events. Developing a deeper understanding of HCV entry will clearly take some time given the complexity of the process and the current state of affairs. Nevertheless, interfering with HCV entry holds promise for drug design and discovery as the mechanistic insights emerge and coalesce into a coherent biochemical description of the process.

Topics: Drug Delivery Systems; Hepacivirus; Hepatitis C; Humans; Membrane Fusion; Viral Envelope Proteins

Topics: Antibody Formation; Hepacivirus; Hepatitis C; Humans; Mutation; Viral Envelope Proteins; Viral Proteins

Topics: Animals; Antigens, CD; Cell Adhesion Molecules; Hepacivirus; Hepatitis C; Humans; Lectins, C-Type; Receptors, Cell Surface; Receptors, Immunologic; Receptors, LDL; Receptors, Scavenger; Receptors, Virus; Tetraspanin 28; Viral Envelope Proteins

Topics: Antiviral Agents; Drug Resistance, Viral; Drug Therapy, Combination; Genotype; Hepacivirus; Hepatitis C; Humans; Interferons; Mutation; Ribavirin; Treatment Outcome; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins

Topics: Animals; Clinical Trials as Topic; Cross Reactions; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoglobulin G; Viral Envelope Proteins; Viral Proteins; Viral Vaccines

Topics: Animals; Glycoproteins; Hepacivirus; Hepatitis C; Humans; Pan troglodytes; Recombinant Proteins; Vaccination; Vaccines, DNA; Viral Envelope Proteins; Viral Hepatitis Vaccines

Hepatitis C virus (HCV) is an emerging virus of great medical significance. A low drug-response rate and a high frequency of persistent infection have caused HCV to reach pandemic proportions. Many infected individuals go on to develop liver cirrhosis and hepatocellular carcinoma, and HCV is now the leading reason for liver transplants in the United States. Differences in genotype response to interferon therapy suggests that one or more viral genes may participate in evasion of the interferon-mediated cellular antiviral response. This review focuses on the viral genes that interact with the host cell to evade the interferon response and on the insights that these interactions may provide into HCV pathogenesis.

Topics: Antiviral Agents; eIF-2 Kinase; Genes, Viral; Hepacivirus; Hepatitis C; Humans; Interferons; Viral Envelope Proteins; Viral Nonstructural Proteins

Topics: Genetic Variation; Hepacivirus; Hepatitis C; Humans; Viral Envelope Proteins; Viral Hepatitis Vaccines

Rapid allograft infection complicates liver transplantation (LT) in patients with hepatitis C virus (HCV). Pegylated interferon-α and ribavirin therapy after LT has significant toxicity and limited efficacy. The effect of a human monoclonal antibody targeting the HCV E2 glycoprotein (MBL-HCV1) on viral clearance was examined in a randomized, double-blind, placebo-controlled pilot study in patients infected with HCV genotype 1a undergoing LT. Subjects received 11 infusions of 50 mg/kg MBL-HCV1 (n=6) or placebo (n=5) intravenously with three infusions on day of transplant, a single infusion on days 1 through 7 and one infusion on day 14 after LT. MBL-HCV1 was well-tolerated and reduced viral load for a period ranging from 7 to 28 days. Median change in viral load (log10 IU/mL) from baseline was significantly greater (p=0.02) for the antibody-treated group (range -3.07 to -3.34) compared to placebo group (range -0.331 to -1.01) on days 3 through 6 posttransplant. MBL-HCV1 treatment significantly delayed median time to viral rebound compared to placebo treatment (18.7 days vs. 2.4 days, p<0.001). As with other HCV monotherapies, antibody-treated subjects had resistance-associated variants at the time of viral rebound. A combination study of MBL-HCV1 with a direct-acting antiviral is underway.

Topics: Aged; Antibodies, Monoclonal; Biopsy; Double-Blind Method; Female; Genotype; Hepacivirus; Hepatitis C; Humans; Liver; Liver Transplantation; Male; Middle Aged; Pilot Projects; RNA, Viral; Time Factors; Viral Envelope Proteins

The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1-6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1-6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418-446 and aa611-616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434-446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410-425. The isolation of four HC-84 HMAbs binding to the peptide, aa434-446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is relevant for vaccine design for this highly diverse virus.

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Antibodies, Viral; Antigens, Viral; Epitope Mapping; Epitopes; Female; Genotype; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Male; Mutation; Viral Envelope Proteins; Viral Hepatitis Vaccines; Virus Internalization

We have completed a phase 1 safety and immunogenicity trial with hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, with MF59 adjuvant as a candidate vaccine. Neutralizing activity to HCV genotype 1a was detected in approximately 25% of the vaccinee sera. In this study, we evaluated vaccinee sera from poor responders as a potential source of antibody dependent enhancement (ADE) of HCV infection. Sera with poor neutralizing activity enhanced cell culture grown HCV genotype 1a or 2a, and surrogate VSV/HCV pseudotype infection titer, in a dilution dependent manner. Surrogate pseudotypes generated from individual HCV glycoproteins suggested that antibody to the E2 glycoprotein; but not the E1 glycoprotein, was the principle target for enhancing infection. Antibody specific to FcRII expressed on the hepatic cell surface or to the Fc portion of Ig blocked enhancement of HCV infection by vaccinee sera. Together, the results from in vitro studies suggested that enhancement of viral infectivity may occur in the absence of a strong antibody response to HCV envelope glycoproteins.

Topics: Antibodies, Neutralizing; Antibody-Dependent Enhancement; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunity, Humoral; Polysorbates; Squalene; Viral Envelope Proteins; Viral Hepatitis Vaccines

The dynamic features of three specific anti-hepatitis C virus (HCV) antibody subpopulations directed against different conformational epitopes of the viral E2 protein (HCV/E2) have been evaluated in patients with primary and persistent HCV infection; the three subpopulations are present in patients infected with different HCV genotypes and have shown a different activity using a pseudovirus neutralization assay (antibodies e301 and e137 exhibiting high neutralizing activity, while antibody e509 enhancement of HCV infectivity). In sequential samples from five patients with primary HCV infection and different virological outcome, all samples tested negative with the single exception of the e509 antibody in a patient not clearing the virus. In sequential samples from 28 patients with persistent infection under treatment with pegylated interferon-alpha plus ribavirin (14 sustained virological responders and 14 non-responders), the therapy did not selectively influence titers of the two neutralizing antibody subpopulations; otherwise, a net increase of the e509 antibody subpopulation related to enhancement of HCV infectivity was observed in non-responders, but not in sustained virological responders (P = 0.0156). This increase was not related to the trend of total anti-HCV/E2 response. The data indicate that a specific antibody response against these epitopes is elicited only late during the infection, thus not influencing virus clearance during primary infection, and that a selective increase of the antibody subpopulation enhancing virus infectivity is observed only in the cohort of patients not responding to antiviral therapy.

Topics: 5' Untranslated Regions; Adult; Antibody Specificity; Antiviral Agents; Disease Progression; Drug Therapy, Combination; Epitopes; Female; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Interferon-alpha; Male; Middle Aged; Molecular Conformation; Neutralization Tests; Polyethylene Glycols; Ribavirin; Time Factors; Treatment Outcome; Viral Core Proteins; Viral Envelope Proteins; Viremia

The hepatitis C virus (HCV) envelope protein 2 (E2) interacts in vitro with the interferon alpha (IFN-alpha)-inducible double-stranded RNA-activated protein kinase, suggesting a possible mechanism by which HCV may evade the antiviral effects of IFN-alpha. Variability in the part of the HCV E2 gene encoding the carboxy-terminal part of the protein, which includes the interaction domain (E2-PePHD), was explored in 25 patients infected with HCV genotype 1b and receiving IFN-alpha therapy. PCR products were generated and sequenced for 15 patients with a sustained response and for 10 patients with no virological response after treatment with IFN-alpha and ribavirin. PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment, after 2 months in responders, and after 6 months in nonresponders. Quasispecies analysis of the pretreatment PePHD region was performed for isolates from patients displaying amino acid substitutions in this domain on direct sequencing. The E2-PePHD sequence was highly conserved in both resistant and susceptible genotype 1b strains and was identical to the prototype HCV type J sequence. No significant emergence of PePHD mutants during therapy was observed in our clonal analysis, and sporadic mutations and treatment outcomes were not found to be correlated. The PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in HCV type 1b-infected patients.

Topics: Amino Acid Sequence; Antiviral Agents; Drug Therapy, Combination; Genotype; Hepacivirus; Hepatitis C; Humans; Interferon-alpha; Molecular Sequence Data; Mutation; Ribavirin; Sequence Analysis, DNA; Treatment Outcome; Viral Envelope Proteins

In view of the high rate of chronicity of acute hepatitis C and the low efficacy of interferon (IFN) treatment in advanced liver disease, it may be beneficial to treat patients during the acute phase of the infection. Here we assessed the effects of variable-dose IFNalpha-2b treatment in haemodialysis patients with acute hepatitis C virus (HCV) infection, and identified factors that may predict response to this therapy. The study population included 67 patients, but 14 were excluded due to side-effects or because they were lost to follow-up. Seventeen patients who received no specific treatment were used as controls (Group 1). Sixteen and 20 patients received low-(3 MU) and high-dose (6-10 MU) IFNalpha-2b three times weekly for 3 months (Groups 2 and 3, respectively). Virological end-of-treatment response (ETR) was observed in 1 (5.6%), 13 (56.5%), and 17 (65.4%) patients in Groups 1, 2, and 3, respectively, and virological sustained response (SR) was observed in 1 (5.6%), 6 (26.1%), and 13 (50%) patients in the three groups. The rates of virological ETR and SR in the treated groups were significantly higher than those of the control group (P < 0.01 for all comparisons). In multivariate logistic regression analysis, single stranded confirmational polymorphysm (SSCP) band number (P=0.02) was the only factor that was significantly associated with virological SR. In conclusion, IFN-alpha treatment initiated during the acute phase of HCV infection is associated with a higher rate of virological ETR and SR. This study suggested that quasispecies heterogeneity has predictive value with regard to virological SR.

Topics: 5' Untranslated Regions; Acute Disease; Adult; Antiviral Agents; Female; Genotype; Hepacivirus; Hepatitis C; Humans; Interferon alpha-2; Interferon-alpha; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Predictive Value of Tests; Recombinant Proteins; Renal Dialysis; RNA, Viral; Treatment Outcome; Viral Envelope Proteins

The extremely high rate of chronicity to hepatitis C virus (HVC) infection suggests an inefficient immune response. The humoral immune response to HCV was evaluated in 60 patients with chronic HCV infection and in 12 patients acutely infected with HCV.. A number of recombinant HCV antigens including the core, envelope 2 (E2), nonstructural (NS) 3, NS4, and NS5 proteins, and NS4a and E2-HVR-1 peptides were used in enzyme-linked immunoassays.. Immunoglobulin (Ig) G antibody responses to these viral antigens, except for the HCV core, were highly restricted to the IgG1 isotype. The prevalence of antibodies of the IgG1 isotype specific for the HCV core, E2, E2-HVR1, NS3 (helicase domain), NS4, and NS5 antigens was 97%, 98%, 28%, 88%, 33%, and 68%, respectively. Antibodies of the IgG3 isotype specific for E2, E2-HVR-1, NS3, NS4, and NS5 were detected in a minority of serum samples. The IgG2 and IgG4 isotypes were rarely if ever detected. Furthermore, antibody responses to HCV viral antigens were of relatively low titer and, with the exception of anti-HCV core, were delayed in appearance until the chronic phase of infection.. The IgG1 restriction, low titer, and delayed appearance of antibody responses elicited during HCV infection suggest that the immunogenicity of HCV proteins is limited in the context of natural infection. Inasmuch as recombinant HCV viral antigens perform as relatively normal immunogens in small animals, we suggest that the defective humoral immune responses during HCV infection may be attributable to an "immune avoidance" strategy.

Topics: Acute Disease; Antibody Formation; Enzyme-Linked Immunosorbent Assay; Hepatitis C; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Immunoglobulin G; Kinetics; Viral Core Proteins; Viral Envelope Proteins

To determine the effects of interferon-alpha (IFN-alpha) and ribavirin therapy on hepatitis C virus (HCV) quasispecies heterogeneity, 29 patients with chronic HCV infection treated with either IFN-alpha (n = 15), ribavirin (n = 7) or placebo (n = 7) were studied. HCV quasispecies heterogeneity was determined by single-strand conformational polymorphism (SSCP) analysis of the HCV E2 hypervariable region 1 (HVR1). For patients receiving IFN-alpha, HVR1 was amplified in 14 of 15 patients before, and in six of seven patients after therapy. After controlling the amount of amplicon loaded, a reduction in the number of SSCP bands was observed with IFN-alpha therapy (median number of SSCP bands per patient was eight before therapy and two after therapy). In the seven patients within each of the ribavirin- and placebo-treated groups, there was no significant difference in the viraemia level, number of SSCP bands per patient or the SSCP band pattern, before and after therapy. These findings suggest that at the doses given, IFN-alpha, but not ribavirin, exerts a selective pressure on HCV quasispecies heterogeneity.

Topics: Adult; Aged; Alanine Transaminase; Antiviral Agents; Female; Follow-Up Studies; Genetic Heterogeneity; Hepacivirus; Hepatitis C; Humans; Interferon-alpha; Male; Middle Aged; Polymorphism, Single-Stranded Conformational; Ribavirin; RNA, Viral; Viral Envelope Proteins

Despite the development of highly effective hepatitis C virus (HCV) treatments, an effective prophylactic vaccine is still lacking. HCV infection is mediated by its envelope glycoproteins, E1 and E2, during the entry process, with E2 binding to cell receptors and E1 mediating endosomal fusion. The structure of E1E2 has only been partially resolved by X-ray crystallography of the core domain of E2 protein (E2c) and its complex with various neutralizing antibodies. Structural understanding of the E1E2 heterodimer in its native form can advance the design of candidates for HCV vaccine development. Here, we analyze the structure of the recombinant HCV E1E2 heterodimer with the aid of well-defined monoclonal anti-E1 and E2 antibodies, as well as a small-molecule chlorcyclizine-diazirine-biotin that can target and cross-link the putative E1 fusion domain. Three-dimensional (3D) models were generated after extensive 2D classification analysis with negative-stain single-particle data sets. We modeled the available crystal structures of the E2c and Fabs into 3D volumes of E1E2-Fab complexes based on the shape and dimension of the domain density. The E1E2 heterodimer exists in monomeric form and consists of a main globular body, presumably depicting the E1 and E2 stem/transmembrane domain, and a protruding structure representing the E2c region, based on anti-E2 Fab binding. At low resolution, a model generated from negative-stain analysis revealed the unique binding and orientation of individual or double Fabs onto the E1 and E2 components of the complex. Cryo-electron microscopy (cryo-EM) of the double Fab complexes resulted in a refined structural model of the E1E2 heterodimer, presented here.

Topics: Antibodies, Neutralizing; Cryoelectron Microscopy; Electrons; Hepacivirus; Hepatitis C; Humans; Imaging, Three-Dimensional; Protein Conformation; Viral Envelope Proteins

Antibodies targeting the hepatitis C virus (HCV) envelope glycoprotein E2 are associated with delayed disease progression, and these antibodies can also facilitate spontaneous clearance of infection in some individuals. However, many infected people demonstrate low titer and delayed anti-E2 antibody responses. Since a goal of HCV vaccine development is induction of high titers of anti-E2 antibodies, it is important to define the mechanisms underlying these suboptimal antibody responses. By staining lymphocytes with a cocktail of soluble E2 (sE2) glycoproteins, we detected HCV E2-specific (sE2+) B cells directly ex vivo at multiple acute infection timepoints in 29 HCV-infected subjects with a wide range of anti-E2 IgG titers, including 17 persistently infected subjects and 12 subjects with spontaneous clearance of infection. We performed multi-dimensional flow cytometric analysis of sE2+ and E2-nonspecific (sE2-) class-switched B cells (csBC). In sE2+ csBC from both persistence and clearance subjects, frequencies of resting memory B cells (rMBC) were reduced, frequencies of activated MBC (actMBC) and tissue-like MBC (tlMBC) were increased, and expression of FCRL5, an IgG receptor, was significantly upregulated. Across all subjects, plasma anti-E2 IgG levels were positively correlated with frequencies of sE2+ rMBC and sE2+ actMBC, while anti-E2 IgG levels were negatively correlated with levels of FCRL5 expression on sE2+ rMBC and PD-1 expression on sE2+ actMBC. Upregulation of FCRL5 on sE2+ rMBC and upregulation of PD-1 on sE2+ actMBC may limit anti-E2 antibody production in vivo. Strategies that limit upregulation of these molecules could potentially generate higher titers of protective antibodies against HCV or other pathogens.

Topics: B-Lymphocytes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Programmed Cell Death 1 Receptor; Receptors, Fc; Viral Envelope Proteins

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.

Topics: B-Lymphocytes; Broadly Neutralizing Antibodies; Complementarity Determining Regions; Epitopes; Female; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoglobulin Heavy Chains; Male; Middle Aged; Mutation; Viral Envelope Proteins

Recent studies identified signal peptidase complex subunit 1 (SPCS1) as a proviral host factor for Flaviviridae viruses, including HCV. One of the SPCS1's roles in flavivirus propagation was attributed to its regulation of signal peptidase complex (SPC)-mediated processing of flavivirus polyprotein, especially C-prM junction. However, whether SPCS1 also regulates any SPC-mediated processing sites within HCV polyprotein remains unclear. In this study, we determined that loss of SPCS1 specifically impairs the HCV E2-p7 processing by the SPC. We also determined that efficient separation of E2 and p7, regardless of its dependence on SPC-mediated processing, leads to SPCS1 dispensable for HCV assembly These results suggest that SPCS1 regulates HCV assembly by facilitating the SPC-mediated processing of E2-p7 precursor. Structural modeling suggests that intrinsically delayed processing of the E2-p7 is likely caused by the structural rigidity of p7 N-terminal transmembrane helix-1 (p7/TM1/helix-1), which has mostly maintained membrane-embedded conformations during molecular dynamics (MD) simulations. E2-p7-processing-impairing p7 mutations narrowed the p7/TM1/helix-1 bending angle against the membrane, resulting in closer membrane embedment of the p7/TM1/helix-1 and less access of E2-p7 junction substrate to the catalytic site of the SPC, located well above the membrane in the ER lumen. Based on these results we propose that the key mechanism of action of SPCS1 in HCV assembly is to facilitate the E2-p7 processing by enhancing the E2-p7 junction site presentation to the SPC active site. By providing evidence that SPCS1 facilitates HCV assembly by regulating SPC-mediated cleavage of E2-p7 junction, equivalent to the previously established role of this protein in C-prM junction processing in flavivirus, this study establishes the common role of SPCS1 in Flaviviridae family virus propagation as to exquisitely regulate the SPC-mediated processing of specific, suboptimal target sites.

Topics: Cell Line; HEK293 Cells; Hepacivirus; Hepatitis C; Host Microbial Interactions; Humans; Membrane Proteins; Molecular Dynamics Simulation; Protein Conformation; Viral Envelope Proteins; Viroporin Proteins; Virus Assembly; Virus Replication

SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

Topics: Animals; Broadly Neutralizing Antibodies; Hepatitis C; Hepatitis C Antibodies; Immunogenicity, Vaccine; Mice; Protein Multimerization; Viral Envelope Proteins; Viral Hepatitis Vaccines

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma in humans and afflicts more than 58 million people worldwide. The HCV envelope E1 and E2 glycoproteins are essential for viral entry and comprise the primary antigenic target for neutralizing antibody responses. The molecular mechanisms of E1E2 assembly, as well as how the E1E2 heterodimer binds broadly neutralizing antibodies, remain elusive. Here, we present the cryo-electron microscopy structure of the membrane-extracted full-length E1E2 heterodimer in complex with three broadly neutralizing antibodies-AR4A, AT1209, and IGH505-at ~3.5-angstrom resolution. We resolve the interface between the E1 and E2 ectodomains and deliver a blueprint for the rational design of vaccine immunogens and antiviral drugs.

Topics: Antiviral Agents; Broadly Neutralizing Antibodies; Cryoelectron Microscopy; Hepacivirus; Hepatitis C; Humans; Protein Multimerization; Viral Envelope Proteins; Viral Hepatitis Vaccines

Entry of the hepatitis C virus (HCV) into host cells is a multistep process mediated by several host factors, including a tight junction protein claudin-1 (CLDN1). We repeatedly passaged HCV-JFH1-tau, an HCV substrain with higher infectivity, on Huh7.5.1-8 cells. A multi-passaged HCV-JFH1-tau lot was infectious to CLDN1-defective S7-A cells, non-permissive to original HCV-JFH1-tau infection. We identified a single mutation, M706L, in the E2 glycoprotein of the HCV-JFH1-tau lot as an essential mutation for infectivity to S7-A cells. The pseudovirus JFH1/M706L mutant could not infect human embryonic kidney 293 T (HEK293T) cells lacking CLDN family but infected HEK293T cells expressing CLDN1, CLDN6, or CLDN9. Thus, this mutant virus could utilize CLDN1, and other CLDN6 and CLDN9, making HCV possible to infect cells other than hepatocytes. iPS cells, one of the stem cells, do not express CLDN1 but express CLDN6 and other host factors required for HCV infection. We confirmed that the HCV-JFH1-tau-derived mutant with an M706L mutation infected iPS cells in a CLDN6-dependent manner. These results demonstrated that a missense mutation in E2 could broaden the CLDN member specificity for HCV infection. HCV may change its receptor requirement through a single amino acid mutation and infect non-hepatic cells.

Topics: Claudin-1; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Mutation, Missense; Viral Envelope Proteins

Induction of broadly neutralizing monoclonal antibodies (bNAbs) that bind to the viral envelope glycoproteins is a major goal of hepatitis C virus (HCV) vaccine research. The study of bNAbs arising in natural infection is essential in this endeavor. We generated a human antibody, 8D6, recognizing the E2 protein of HCV isolated from a chronic hepatitis C patient. This antibody shows broadly neutralizing activity, which covers a pan-genotypic panel of cell culture-derived HCV virions (HCVcc). Functional and epitope analyses demonstrated that 8D6 can block the interaction between E2 and CD81 by targeting a highly conserved epitope on E2. We describe how the 8D6 lineage evolved via somatic hypermutation to achieve broad neutralization. We found that the V(D)J recombination-generated junctional and somatic hypermutation-induced disulfide bridge (C-C) motif in the CDRH3 is critical for the broad neutralization and binding activity of 8D6. This motif is conserved among a series of broadly neutralizing HCV antibodies, indicating a common binding model. Next, the 8D6 inferred germline (iGL) was reconstructed and tested for its binding affinity and neutralization activity. Interestingly, 8D6 iGL-mediated relatively strong inhibition of the 1b genotype PR79L9 strain, suggesting that PR79L9 may serve as a potential natural viral strain that provides E2 sequences that induce bNAbs. Overall, our detailed epitope mapping and genetic studies of the HCV E2-specific mAb 8D6 have allowed for further refinement of antigenic sites on E2 and reveal a new mechanism to generate a functional CDRH3, while its iGL can serve as a probe to identify potential HCV vaccine strains.

Topics: Antibodies, Monoclonal; Broadly Neutralizing Antibodies; Complementarity Determining Regions; Epitope Mapping; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoglobulin Heavy Chains; Mutation; Protein Interaction Domains and Motifs; Viral Envelope Proteins

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293T

Topics: Antibody Affinity; Gene Knockdown Techniques; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Mannose; Polysaccharides; Protein Binding; Receptors, Virus; Tetraspanin 28; Viral Envelope Proteins

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Epitope Mapping; Epitopes; Female; Gene Expression; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunogenicity, Vaccine; Mice; Models, Molecular; Protein Binding; Protein Conformation; Protein Engineering; Protein Multimerization; Receptors, Virus; Recombinant Proteins; Solubility; Tetraspanin 28; Vaccination; Viral Envelope Proteins; Viral Hepatitis Vaccines

Topics: Antigens, CD; Biosensing Techniques; Conalbumin; Electrochemical Techniques; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Humans; Ligands; Protein Binding; Viral Envelope Proteins

Hepatitis C virus (HCV) can be cleared naturally in a subset of individuals. However, the asymptomatic nature of acute HCV infection makes the study of the early immune response and defining the correlates of protection challenging. Despite this, there is now strong evidence implicating the humoral immune response, specifically neutralising antibodies, in determining the clearance or chronicity outcomes of primary HCV infection. In general, immunoglobulin G (IgG) plays the major role in viral neutralisation. However, there are limited investigations of anti-HCV envelope protein 2 (E2) isotypes (IgM, IgG, IgA) and IgG subclasses (IgG1-4) in early HCV infection. In this study, using a rare cohort of 14 very recently HCV-infected individuals (4-45 days) with varying disease outcome (

Topics: Acute Disease; Adult; Antibodies, Neutralizing; Antibody Formation; Binding Sites, Antibody; Female; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunity, Humoral; Immunoglobulin G; Immunoglobulin M; Longitudinal Studies; Male; Prospective Studies; Viral Envelope Proteins; Young Adult

In this research we describe the improvement of the water-solubility of cyclic epitope mimics based on the HCV E2 glycoprotein by incorporation of suitable polar hinges. The poor solubility of epitope mimics based on peptide sequences in the envelope (E2) protein hampered their synthesis and purification and made it very difficult to prepare the molecular constructs for evaluation of their bioactivity. Since changes in the amino acid composition are hardly possible in these epitope mimics in order to increase water-solubility, a polar cyclization hinge may offer a remedy leading to a significant increase of polarity and therefore water solubility. These polar hinges were applied in the synthesis of better water-soluble HCV-E2 epitopes. An azide functionality in the polar hinges allowed attachment of a tetraethylene glycol linker by Cu-catalyzed azide-alkyne cyclo-addition (CuAAC) for a convenient conjugation to ELISA plates in order to evaluate the bio-activity of the epitope mimics. The immunoassays showed that the use of more polar cyclization hinges still supported anti-HCV antibody recognition and did not negatively influence their binding. This significantly increased solubility induced by polar hinges should therefore allow for the molecular construction and ultimate evaluation of synthetic vaccine molecules.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Cyclization; Enzyme-Linked Immunosorbent Assay; Epitopes; Hepatitis C; Hepatitis C Antibodies; Humans; Solubility; Vaccines, Synthetic; Viral Envelope Proteins

The glycoproteins of hepatitis C virus, E1E2, are unlike any other viral fusion machinery yet described, and are the current focus of immunogen design in HCV vaccine development; thus, making E1E2 both scientifically and medically important. We used pre-existing, but fragmentary, structures to model a complete ectodomain of the major glycoprotein E2 from three strains of HCV. We then performed molecular dynamic simulations to explore the conformational landscape of E2, revealing a number of important features. Despite high sequence divergence, and subtle differences in the models, E2 from different strains behave similarly, possessing a stable core flanked by highly flexible regions, some of which perform essential functions such as receptor binding. Comparison with sequence data suggest that this consistent behaviour is conferred by a network of conserved residues that act as hinge and anchor points throughout E2. The variable regions (HVR-1, HVR-2 and VR-3) exhibit particularly high flexibility, and bioinformatic analysis suggests that HVR-1 is a putative intrinsically disordered protein region. Dynamic cross-correlation analyses demonstrate intramolecular communication and suggest that specific regions, such as HVR-1, can exert influence throughout E2. To support our computational approach we performed small-angle X-ray scattering with purified E2 ectodomain; this data was consistent with our MD experiments, suggesting a compact globular core with peripheral flexible regions. This work captures the dynamic behaviour of E2 and has direct relevance to the interaction of HCV with cell-surface receptors and neutralising antibodies.

Topics: Antibodies, Neutralizing; Antibodies, Viral; Computer Simulation; Epitopes; Glycosylation; HEK293 Cells; Hepatitis C; Humans; Molecular Dynamics Simulation; Protein Binding; Protein Domains; Scattering, Radiation; Viral Envelope Proteins; Virus Internalization; X-Rays

Topics: Amino Acid Sequence; Binding Sites; Cambodia; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Mutation Rate; Phylogeny; Prevalence; Protein Domains; RNA, Viral; Tetraspanin 28; Viral Envelope Proteins

Hepatitis C virus (HCV) infection remains a major worldwide health problem despite development of highly effective direct-acting antivirals. HCV rapidly evolves upon acute infection and generates multiple viral variants (quasispecies), leading to immune evasion and persistent viral infection. Identification of epitopes of broadly neutralizing anti-HCV antibodies (nAbs) is critical to guide HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Epitopes; Evolution, Molecular; Genotype; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immune Evasion; Neutralization Tests; Quasispecies; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins; Viral Structural Proteins

The E2 envelope glycoprotein of the hepatitis C virus (HCV) is a major target of broadly neutralizing antibodies that are closely related to a spontaneous cure of HCV infection. There is still no data about the diversity of E2-specific antibodies (Abs) glycosylation. The aim of this study was to analyze the level and sialylation of E2 IgG Abs, the relation of the respective changes to hepatic fibrosis (F) progression and their possible association with the efficacy of interferon-

Topics: Adult; Antibodies, Viral; Antiviral Agents; Genotype; Glycosylation; Hepacivirus; Hepatitis C; Humans; Immunoglobulin G; Interferon alpha-2; Liver Cirrhosis; Middle Aged; Ribavirin; Treatment Outcome; Viral Envelope Proteins; Viral Load; Young Adult

An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of broadly neutralizing antibodies (bNAbs) in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antibody Formation; Antigens, Viral; Cell Line; Epitopes; Female; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunogenicity, Vaccine; Mice; Models, Molecular; Neutralization Tests; Protein Conformation; Viral Envelope Proteins; Viral Hepatitis Vaccines

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Cell Line; Complementarity Determining Regions; Epitopes; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Neutralization Tests; Receptors, Scavenger; Scavenger Receptors, Class B; Vaccines, Synthetic; Viral Envelope Proteins; Viral Hepatitis Vaccines

Induction of cross-reactive antibodies targeting conserved epitopes of the envelope proteins E1E2 is a key requirement for an hepatitis C virus vaccine. Conserved epitopes like the viral CD81-binding site are targeted by rare broadly neutralizing antibodies. However, these viral segments are occluded by variable regions and glycans. We aimed to identify antigens exposing conserved epitopes and to characterize their immunogenicity.. We created hepatitis C virus variants with mutated glycosylation sites and/or hypervariable region 1 (HVR1). Exposure of the CD81 binding site and conserved epitopes was quantified by soluble CD81 and antibody interaction and neutralization assays. E2 or E1-E2 heterodimers with mutations causing epitope exposure were used to immunize mice. Vaccine-induced antibodies were examined and compared with patient-derived antibodies.. Mutant viruses bound soluble CD81 and antibodies targeting the CD81 binding site with enhanced efficacy. Mice immunized with E2 or E1E2 heterodimers incorporating these modifications mounted strong, cross-binding, and non-interfering antibodies. E2-induced antibodies neutralized the autologous virus but they were not cross-neutralizing.. Viruses lacking the HVR1 and selected glycosylation sites expose the CD81 binding site and cross-neutralization antibody epitopes. Recombinant E2 proteins carrying these modifications induce strong cross-binding but not cross-neutralizing antibodies.. Conserved viral epitopes can be made considerably more accessible for binding of potently neutralizing antibodies by deletion of hypervariable region 1 and selected glycosylation sites. Recombinant E2 proteins carrying these mutations are unable to elicit cross-neutralizing antibodies suggesting that exposure of conserved epitopes is not sufficient to focus antibody responses on production of cross-neutralizing antibodies.

Topics: Animals; Binding Sites; Broadly Neutralizing Antibodies; Cell Line, Tumor; Cross Reactions; Epitopes; Gene Deletion; Glycosylation; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Mice; Mice, Inbred BALB C; Receptors, Virus; Tetraspanin 28; Vaccination; Viral Envelope Proteins; Viral Proteins; Viral Vaccines

In the sera of infected patients, hepatitis C virus (HCV) particles display heterogeneous forms with low-buoyant densities (<1.08), underscoring their lipidation via association with apoB-containing lipoproteins, which was proposed to occur during assembly or secretion from infected hepatocytes. However, the mechanisms inducing this association remain poorly-defined and most cell culture grown HCV (HCVcc) particles exhibit higher density (>1.08) and poor/no association with apoB. We aimed to elucidate the mechanisms of lipidation and to produce HCVcc particles resembling those in infected sera.. We produced HCVcc particles of Jc1 or H77 strains from Huh-7.5 hepatoma cells cultured in standard conditions (10%-fetal calf serum) vs. in serum-free or human serum conditions before comparing their density profiles to patient-derived virus. We also characterized wild-type and Jc1/H77 hypervariable region 1 (HVR1)-swapped mutant HCVcc particles produced in serum-free media and incubated with different serum types or with purified lipoproteins.. Compared to serum-free or fetal calf serum conditions, production with human serum redistributed most HCVcc infectious particles to low density (<1.08) or very-low density (<1.04) ranges. In addition, short-time incubation with human serum was sufficient to shift HCVcc physical particles to low-density fractions, in time- and dose-dependent manners, which increased their specific infectivity, promoted apoB-association and induced neutralization-resistance. Moreover, compared to Jc1, we detected higher levels of H77 HCVcc infectious particles in very-low-density fractions, which could unambiguously be attributed to strain-specific features of the HVR1 sequence. Finally, all 3 lipoprotein classes, i.e., very-low-density, low-density and high-density lipoproteins, could synergistically induce low-density shift of HCV particles; yet, this required additional non-lipid serum factor(s) that include albumin.. The association of HCV particles with lipids may occur in the extracellular milieu. The lipidation level depends on serum composition as well as on HVR1-specific properties. These simple culture conditions allow production of infectious HCV particles resembling those of chronically-infected patients.. Hepatitis C virus (HCV) particles may associate with apoB and acquire neutral lipids after exiting cells, giving them low-buoyant density. The hypervariable region 1 (HVR1) is a majorviral determinant of E2 that controls this process. Besides lipoproteins, specific serum factors including albumin promote extracellular maturation of HCV virions. HCV particle production in vitro, with media of defined serum conditions, enables production of infectious particles resembling those of chronically infected patients.

Topics: Apolipoprotein B-100; Apolipoproteins E; Cell Line, Tumor; Cholesterol, HDL; Cholesterol, LDL; Cholesterol, VLDL; Culture Media, Conditioned; Culture Media, Serum-Free; Extracellular Fluid; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Serum Albumin, Human; Viral Envelope Proteins; Viral Proteins; Virion; Virus Assembly

This chapter describes how to generate chimeric molecular cassettes that are ready to receive PCR-amplified E1/E2 genes using new DNA cloning technology. The method is divided into three sections: (1) generation of a ΔCore-NS2 cassette based upon the full-length JFH-1 molecular clone; (2) insertion of a "structural gene" fragment encoding the Core, p7, and NS2 genes of a given genotype reference sequence, to generate a ΔE1/E2 cassette; and (3) insertion of patient-isolated E1/E2 genes that are genotype-matched to the structural genes. The final assembled chimeric genomes can then be analyzed in the HCV cell culture system. These cassettes allow characterization of the extensive in vivo viral diversity without the need to isolate and clone whole virus genomes. This method can be readily applied to the study of other HCV genes and other viruses.

Topics: Cloning, Molecular; Escherichia coli; Genes, Viral; Genotype; Hepacivirus; Hepatitis C; Humans; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Viral Envelope Proteins; Viral Nonstructural Proteins

Experimental characterization of the properties of authentic viruses circulating in infected individuals presents a problem when investigating RNA viruses with error-prone polymerases. The hepatitis C virus provides an extreme example of RNA virus genetic variability, as the nucleotide composition of HCV genomes can vary by more than 30% between strains. The envelope glycoproteins E1 and E2 in particular are able to tolerate a particularly high level of variation. They are under continual selection pressure from the host antibody response during chronic infection and can tolerate adaptive mutations, leading to great diversity in a single host. The diversity of E1/E2 in circulating viruses has hindered investigations of their function and development of a vaccine that will generate antibodies able to potently neutralize entry of genetically distinct strains.Here we describe methods used in our laboratory to overcome the limitations of investigating the properties of the envelope glycoproteins representing only small numbers of HCV variants. Using a high-fidelity, limiting dilution ("endpoint") PCR approach to amplify single E1/E2 cDNA templates, which can then generate recombinant model viral particles using retrovirus packaging/reporter constructs. These retroviral pseudoparticles (pseudotypes) facilitate investigation of the properties of authentic E1/E2 glycoproteins in a single-round infection assay. We also describe optimized methods for generation of infectious pseudoparticles from patient-isolated E1/E2 and methods for performing neutralization assays with both anti-virus and anti-host antibodies.

Topics: Base Sequence; Cloning, Molecular; DNA, Complementary; Genes, Viral; Genetic Vectors; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Polymerase Chain Reaction; Viral Envelope Proteins; Virion

The hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, are crucial for HCV assembly and entry, and are promising vaccine antigens. However, they are challenging to study because of technical difficulties in protein production and in quality control for protein folding and glycosylation. To study E1 and E2 in different experimental systems, e.g. infected cells, virus culture, virus-like particles, and clinical samples, a standardized method to accurately quantify the glycoproteins will be essential for most research projects. Here we outline a sensitive assay based on dual-color fluorescence immunoblot and the Odyssey imaging system to detect and quantify HCV E1 and E2 glycoproteins either using a purified E1E2 complex, or an engineered protein standard containing E1 and E2 at equal molar ratio. The method is capable of simultaneously detecting and quantifying as little as 7 ng of E1 and 5 ng of E2 in HCV pseudoparticles, and will be useful to quantify E1 and E2 from a wide variety of samples.

Topics: Animals; Cell Line; Fluorescence; Hepacivirus; Hepatitis C; Humans; Immunoblotting; Viral Envelope Proteins

Posttranslational modifications (PTMs) are often required for proper folding and physiological function of proteins, including the envelope glycoproteins 1 and 2 (E1 and E2) of hepatitis C virus (HCV). Commonly used expression systems such as bacteria, yeast, and baculovirus produce soluble HCV E1 and E2 at very low yields, as the cellular environment and molecular machinery necessary for PTMs may be suboptimal or missing. Here, we describe an expression system for HCV E2 ectodomain (eE2) with 11 N-linked glycans and eight disulfide bonds, which combines lentivirus transduction of mammalian cells and a continuous growth, adherent cell bioreactor. It is environmentally friendly, as well as cost- and time-efficient compared to other methods of recombinant protein expression in mammalian systems with final yields of eE2 approaching 60 mg/L of cell culture supernatant. eE2 produced by this system is amenable to a variety of biophysical studies, including structural determination by X-ray crystallography. Considering the ease of use and flexibility, this method can be applied to express an array of difficult target proteins in a variety of mammalian cell lines.

Topics: Animals; Bioreactors; Cell Line; CHO Cells; Cloning, Molecular; Cricetulus; Gene Expression; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Lentivirus; Plasmids; Protein Domains; Protein Processing, Post-Translational; Recombinant Proteins; Transduction, Genetic; Viral Envelope Proteins

The envelope glycoproteins E1 and E2 of hepatitis C virus form a heterodimeric complex on the viral surface. They are the targets of neutralizing antibodies and are being investigated as potential vaccine antigens. Because of the high level of cysteine residues and N-glycosylation sites in the polypeptide sequences, it is technically challenging to produce pure, folded recombinant E1, E2, and E1E2 complex for downstream analysis. In this chapter, the methods we used to isolate a panel of human antibodies specific to diverse antigenic regions on the glycoproteins are discussed. The antibodies have been found to be valuable reagents for the study of HCV envelope glycoproteins, including the determination of the first E2 core domain structure.

Topics: Bacteriophages; Cell Surface Display Techniques; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunogenicity, Vaccine; Protein Domains; Viral Envelope Proteins; Viral Hepatitis Vaccines

Enzyme-linked immunosorbent assays (ELISAs) enable rapid detection and quantitation of antibodies in samples. Such assays can be highly sensitive and can be performed in most laboratories with basic equipment. Although detecting binding antibodies to the surface proteins of most pathogens by ELISA is not always indicative of antibody function, i.e., neutralizing activity of antibodies, the results can be used as a first step toward more in-depth analysis of antibody responses. Here we describe a method that can be used to standardize ELISAs for the detection of HCV envelope antibodies across laboratories and provide adaptations of the method to further characterize antibody responses in serum samples.

Topics: Animals; Antibodies, Neutralizing; Cell Line; Cricetulus; Enzyme-Linked Immunosorbent Assay; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Mannose-Binding Lectins; Neutralization Tests; Plant Lectins; Viral Envelope Proteins

Hepatitis C virus (HCV) pseudoparticles (HCVpp) are generated by cotransfection of HCV envelope (E1 and E2) genes along with a retroviral packaging/reporter construct into HEK293T cells. Enveloped particles bearing HCV E1E2 proteins on their surface are released through a retroviral budding process into the supernatant. Viral E1E2 glycoproteins facilitate a single round of receptor-mediated entry of HCVpp into hepatoma cells, which can be quantified by reporter gene expression. These HCVpp have been employed to study mechanisms of HCV entry into hepatoma cells, as well as HCV neutralization by immune sera or HCV-specific monoclonal antibodies.

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Cell Line, Tumor; Genes, Reporter; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Neutralization Tests; Viral Envelope Proteins; Virion; Virus Internalization; Virus Release

Despite the development of direct-acting antivirals (DAAs), hepatitis C virus (HCV) infection remains a major cause for liver disease and cancer worldwide. Entry inhibitors block virus host cell entry and, therefore, prevent establishment of chronic infection and liver disease. Due to their unique mechanism of action, entry inhibitors provide an attractive antiviral strategy in organ transplantation. In this study, we developed an innovative approach in preventing HCV infection using a synergistic combination of a broadly neutralizing human monoclonal antibody (HMAb) targeting the HCV E2 protein and a host-targeting anti-claudin 1 (CLDN1) humanized monoclonal antibody. An in vivo proof-of-concept study in human liver-chimeric FRG-NOD mice proved the efficacy of the combination therapy at preventing infection by an HCV genotype 1b infectious serum. While administration of individual antibodies at lower doses only showed a delay in HCV infection, the combination therapy was highly protective. Furthermore, the combination proved to be effective in preventing infection of primary human hepatocytes by neutralization-resistant HCV escape variants selected during liver transplantation, suggesting that a combination therapy is suited for the neutralization of difficult-to-treat variants. In conclusion, our findings suggest that the combination of two HMAbs targeting different steps of virus entry improves treatment efficacy while simultaneously reducing treatment duration and costs. Our approach not only provides a clinical perspective to employ HMAb combination therapies to prevent graft re-infection and its associated liver disease but may also help to alleviate the urgent demand for organ transplants by allowing the transplantation of organs from HCV-positive donors.

Topics: Animals; Animals, Genetically Modified; Antibodies, Monoclonal; Antibodies, Neutralizing; Antiviral Agents; Claudin-1; Drug Combinations; Drug Synergism; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Mice, Inbred NOD; Proof of Concept Study; Viral Envelope Proteins; Virus Internalization

An effective vaccine to the antigenically diverse hepatitis C virus (HCV) must target conserved immune epitopes. Here, we investigate cross-neutralization of HCV genotypes by broadly neutralizing antibodies (bNAbs) encoded by the relatively abundant human gene family

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Antibody Affinity; Binding Sites; Blood Donors; Cell Line, Tumor; Cross Reactions; Dual-Specificity Phosphatases; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Viral Envelope Proteins; Viral Hepatitis Vaccines

Hepatitis C Virus is a blood borne pathogen responsible for chronic hepatitis in more than 71 million people. Wide variations across strains and genotypes are one of the major hurdles in therapeutic development. While genotype 1 remains the most extensively studied and abundant strain, genotype 3 is more virulent and second most prevalent. This study aimed to compare differences in the glycoprotein E2 across HCV genotypes at nucleotide, protein and structural levels. Nucleotide sequences of E2 from 29 strains across genotypes 1a, 1b, 3a and 3b revealed a stark preference for C-richness which was attributed to a distinct bias for C-rich codons in genotype 1. Genotype 3 exhibited a similar preference to a lesser extent. Amino acid level comparison revealed majority of the changes at the C-terminal half of the proteins leaving the N-terminal region conspicuously conserved apart from the two hyper variable regions. Amino acid changes across genotypes were mostly polar-nonpolar alterations. In silico models of E2 glycoproteins and docking analysis with the energy minimized PDB-CD81 model revealed unique interacting residues in both E2 and CD81. While several CD81 binding residues were common for all four genotypes, number and composition of interacting residues varied. The interacting residues of E2 were however unique for each genotype. E2 of genotype 3a and CD81 had the strongest interaction. In conclusion this is the first comprehensive study comparing E2 sequences across genotypes 1a, 1b, 3a and 3b revealing stark genotype-specific differences which requires more extensive investigation.

Topics: Amino Acids; Binding Sites; Codon; Evolution, Molecular; Genotype; Hepacivirus; Hepatitis C; Host-Pathogen Interactions; Humans; Models, Molecular; Protein Binding; Protein Conformation; Selection, Genetic; Structure-Activity Relationship; Tetraspanin 28; Viral Envelope Proteins; Virus Internalization

The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predicted

Topics: Animals; Antibodies, Neutralizing; Antibody Formation; Cell Line; Epitopes; Female; Glycosylation; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Insecta; Mammals; Mice; Polysaccharides; Sf9 Cells; Viral Envelope Proteins

Despite the advent of direct-acting antivirals (DAAs), HCV remains an important public health problem globally. There is at present no effective vaccine against the virus, and the DAAs in current use cannot prevent de novo infection, including in liver transplant setting wherein donor livers inevitably become re-infected. Developing inhibitors to HCV entry using nature-derived small molecules may help to expand/complement the current treatment options.. In this study, we explored the effect of the plant alkaloid berberine (BBR) on HCV early viral entry.. Cell culture-derived HCV (HCVcc), viral pseudoparticles bearing HCV glycoproteins (HCVpp), and entry-related assays were employed to assess BBR's bioactivity. Molecular docking was used to predict BBR-HCV glycoproteins interaction, and the compound's antiviral activity was confirmed against HCVcc infection of primary human hepatocytes (PHHs).. BBR specifically impeded HCVcc attachment and entry/fusion steps without inactivating the free virus particles or affecting the expression of host cell entry factors and post-entry viral replication. BBR also effectively inhibited infection by viral pseudoparticles expressing HCV E1/E2 glycoproteins and molecular docking analysis pointed at potential interaction with HCV E2. Finally, BBR could suppress HCVcc infection of PHHs.. We identified BBR as a potent HCV entry inhibitor, which merits further evaluation particularly for use in transplant setting against graft re-infection by HCV.

Topics: Antiviral Agents; Berberine; Cells, Cultured; Hepacivirus; Hepatitis C; Hepatocytes; Host-Pathogen Interactions; Humans; Molecular Docking Simulation; Molecular Targeted Therapy; Viral Envelope Proteins; Virus Internalization; Virus Replication

Isolation of broadly neutralizing human monoclonal antibodies (HmAbs) targeting the E2 glycoprotein of Hepatitis C virus (HCV) has sparked hope for effective vaccine development. Nonetheless, escape mutations have been reported. Ideally, a potent vaccine should elicit HmAbs that target regions of E2 that are most difficult to escape. Here, aimed at addressing this challenge, we develop a predictive in-silico evolutionary model for E2 that identifies one such region, a specific antigenic domain, making it an attractive target for a robust antibody response. Specific broadly neutralizing HmAbs that appear difficult to escape from are also identified. By providing a framework for identifying vulnerable regions of E2 and for assessing the potency of specific antibodies, our results can aid the rational design of an effective prophylactic HCV vaccine.

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Antibodies, Viral; Computer Simulation; Drug Design; Epitope Mapping; Epitopes; Evolution, Molecular; Hepacivirus; Hepatitis C; Humans; Models, Biological; Viral Envelope Proteins; Viral Hepatitis Vaccines

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1-6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1-6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.

Topics: Adult; Amino Acid Sequence; Antibodies, Monoclonal; Antibodies, Neutralizing; Drug Design; Epitope Mapping; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Neutralization Tests; Prospective Studies; Sequence Homology; Viral Envelope Proteins; Viral Hepatitis Vaccines; Young Adult

Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV.

Topics: Antibodies, Neutralizing; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Protein Conformation; Protein Multimerization; Receptors, Cell Surface; Recombinant Fusion Proteins; Viral Envelope Proteins; Virus Internalization

The phosphatidylserine-specific phospholipase A1 (PLA1A) is an essential host factor in hepatitis C virus (HCV) assembly. In this study, we mapped the E2, NS2 and NS5A involved in PLA1A interaction to their lumenal domains and membranous parts, through which they form oligomeric protein complexes to participate in HCV assembly. Multiple regions of PLA1A were involved in their interaction and complex formation. Furthermore, the results represented structures with PLA1A and E2 in closer proximity than NS2 and NS5A, and strongly suggest PLA1A-E2's physical interaction in cells. Meanwhile, we mapped the NS5A sequence which participated in PLA1A interaction with the C-terminus of domain 1. Interestingly, these amino acids in the sequence are also essential for viral RNA replication. Further experiments revealed that these four proteins interact with each other. Moreover, PLA1A expression levels were elevated in livers from HCV-infected patients. In conclusion, we exposed the structural determinants of PLA1A, E2, NS2 and NS5A proteins which were important for HCV assembly and provided a detailed characterization of PLA1A in HCV assembly.

Topics: Cell Line, Tumor; Hepacivirus; Hepatitis C; Host-Pathogen Interactions; Humans; Liver; Phosphatidylserines; Phospholipases A1; RNA, Viral; Viral Envelope Proteins; Viral Nonstructural Proteins; Virus Assembly

Hepatitis C (HCV) is a rapidly mutating RNA virus, with a strong propensity to cause chronic infection and progressive liver disease. Recent evidence has shown that early appearance of neutralizing antibodies in primary infection is associated with clearance. Little is known about the characteristics of HCV-specific B cells and their correlation with outcomes in primary infection, as there is a lack of sensitive tools for HCV-specific B cells which are present at very low frequency. We describe the development and optimisation of tetramer staining for flow cytometric detection of HCV-specific B cells using a cocktail of two recombinant HCV Envelope-2 (rE2) glycoproteins (from genotype 1a and 3a; Gt1a and Gt3a) and streptavidin dyes. The optimal weight to weight (w/w) ratio of streptavidin-phycoerythrin (PE) and rE2 proteins were determined for sensitive detection using HCV E2-specific hybridoma cell lines and peripheral blood mononuclear cells (PBMC) from HCV-infected individuals. In a cross-sectional set of PBMC samples collected from 33 subjects with either chronic infection or previous clearance, HCV E2-specific B cells (CD19

Topics: B-Lymphocytes; Cross-Sectional Studies; Female; Flow Cytometry; Genotype; Hepacivirus; Hepatitis C; Humans; Immunologic Memory; Male; Viral Envelope Proteins

Peripheral neuropathy (PN), the major neurological complication of chronic HCV infection, is frequently associated with mixed cryoglobulinaemia (MC) and small-vessel systemic vasculitis. While humoral and cell-mediated immune mechanisms are suspected to act together in an aberrant immune response that results in peripheral nerve damage, the role of HCV remains largely speculative. The possible demonstration of HCV in peripheral nerve tissue would obviously assume important pathogenic implications.. We studied sural nerve biopsies from 11 HCV-positive patients with neuropathic symptoms: five with and six without MC. In situ hybridization (ISH) and immunofluorescence studies were carried out to detect genomic and antigenomic HCV RNA sequences and HCV-encoded E2-glycoprotein, respectively.. Epineurial vascular deposits of E2-glycoprotein were found in four (80%) MC and in two (33.3%) non-MC patients, respectively. These findings were enhanced by the perivascular deposition of positive-, though not negative-strand replicative RNA, as also found in the nerve extracts of all patients. Mild inflammatory cell infiltrates with no deposits of immunoglobulins and/or complement proteins were revealed around small vessels, without distinct vasculitis changes between MC and non-MC patients.. These results indicate that nerve vascular HCV RNA/E2 deposits associated to perivascular inflammatory infiltrates were similar in chronically HCV-infected patients, regardless of cryoglobulin occurrence. Given the failure to demonstrate HCV productive infection in the examined sural nerve biopsies, nerve damage is likely to result from virus-triggered immune-mediated mechanisms.

Topics: Aged; Base Sequence; Biopsy; Female; Hepacivirus; Hepatitis C; Humans; Male; Middle Aged; Peripheral Nervous System Diseases; Sural Nerve; Viral Envelope Proteins

HCV E2 glycoprotein is one of the most attractive proteins for designing an effective vaccine. Deletion of hydrophobic carboxyl-terminal region of this protein is necessary for its secretion, especially when it is expressed in E-coli. In this study we expressed this protein in truncated form and evaluated its application in developing an ELISA test and induction of humoral response in immunized mice.. The purpose of this study was expression of HCV truncated E2 protein from JFH1 strain in E-coli BL21(DE3) and evaluation of its antigenicity.. Truncated E2 region from HCV genotype 2a (JFH1) was amplified by PCR and cloned into a pET28a (+) vector and was used to transform the E-coli DH5α strain. The recombinant E2 protein was evaluated both in an ELISA test and induction of humoral immunity in mice.. Truncated E2 protein was expressed in BL21(DE3). Its specific antibody was detected in serum samples from HCV infected patients. Also, it could elicit a significant humoral immunity in mice.. Truncated form of E2 protein which has been expressed in E-coli could be used as an effective antigen both in diagnostic tests such as ELISA and also, as a protein-based vaccine candidate.

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Female; Gene Expression; Hepacivirus; Hepatitis C; Humans; Immunity, Humoral; Immunization; Mice; Mice, Inbred BALB C; Recombinant Proteins; Vaccines, Synthetic; Viral Envelope Proteins; Viral Hepatitis Vaccines

The high mutation rate of the hepatitis C virus (HCV) genome increases the genotype diversity and renders the detection of the virus more difficult. Therefore, prediction and assessment of highly conserved and strongly antigenic epitope polypeptide sequences have become a focus of current research. The E2 region is the target binding region of neutralizing antibodies. HCV genomics, especially the high mutation rate of E2 region sequence, makes its genotyping more and more diverse, and the detection of HCV and genotype is becoming more and more strict. In this study, four HCV B cell epitope polypeptides were constructed based on assessment of conserved sequences in the HCV E2 region and prediction of B cell epitopes, including sequences specific to genotype 1A (DC-13: 434-DTGWLAGLFYYHK-446), genotype 1B (HC-13: 434-HTGFLAALFYAKS-446), genotype 4D (NC-13: 434-NTGFLASLFYTHK-446), and a consensus sequence (FC-9: 447-FNSSGCPER-455). Epitope polypeptides combined with serum from 29 HCV-infected or 25 non-HCV-infected individuals were assayed by enzyme-linked immunosorbent assay (ELISA), and differences were analyzed by T/T' test methods in SPSS v20.0 software. Binding levels of genotype 1A, 4D, and consensus epitope polypeptides with sera of HCV-infected patients were higher than those of non-infected individuals. Moreover, binding of genotype 1B epitope polypeptides with serum of HCV 1B-infected patients was higher than that of HCV 2A-infected patients. While the screening results of HCV genotype-specific epitope polypeptides were preliminary, these findings indicated that we successfully established an HCV and genotype serological ELISA detection method. Such an approach would facilitate the discovery of epitope polypeptides which may become new antigen candidates in peptide vaccine development for the prevention of HCV infection.

Topics: Adult; Aged; Antibodies, Neutralizing; Conserved Sequence; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Epitopes, B-Lymphocyte; Female; Genotype; Hepacivirus; Hepatitis C; Humans; Immune Sera; Male; Middle Aged; Peptides; Protein Binding; Vaccines, Subunit; Viral Envelope Proteins; Viral Hepatitis Vaccines

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.

Topics: Animals; Carcinoma, Hepatocellular; Evolution, Molecular; Hepacivirus; Hepatitis C; Humans; Liver Neoplasms; Mice; Mice, Inbred C57BL; Protein Binding; Tumor Cells, Cultured; Viral Envelope Proteins; Virus Internalization; Virus Replication

In November 2015, reuse of needles and syringes in conjunction with an increase in cases of HCV at a clinic in Korea was reported and investigated by public health authorities. Patients who received injections at the clinic from the first time this infection control breach may have occurred in 2008 through 2015 when the practice was stopped were offered screening for HCV and other blood-borne pathogens such as HIV, HTLV, HBV, syphilis, and malaria.. The aim of this study was to assess whether an outbreak of hepatitis C had occurred among the potentially exposed clinic patients due to this infection control breach.. We performed hepatitis C viral RNA load tests and genotyping using plasma from hepatitis C antibody-positive individuals who had visited the clinic between May 2008 and November 2015. We analyzed the core-E2 and NS5B regions of the virus from RNA-positive samples by constructing a phylogenetic tree based on maximum likelihood analysis. To identify transmission risk factors and epidemiological relationships among the patients, we reviewed their medical records, assessed staff infection control practices and performed environmental inspection of the clinic. Environmental samples from medication room surfaces and medication vial contents were tested for HCV RNA.. Among the 1721 patients tested, 96 were IgG-positive and 70 were viral RNA-positive. Among the 61 patients whose viral loads were greater than the detection limit, 41 (67.2%) were classified as genotype 1a, 1 (1.6%) as genotype 1b, 18 (29.5%) as genotype 1, and one (1.6%) as genotype 2. After sequencing, 12 genotype 1 cases were further classified as genotype 1a (11) or 1b (1). The sequences of the core-E2 and NS5B regions of 45 patients formed a monophyletic cluster distinct from genotype 1a. The hepatitis C virus sequences from patients and environmental specimens were well-matched in the partial E1 gene region. We detected genotype 1a RNA in environmental specimens, indicating a healthcare-associated outbreak caused by reuse of syringes and contaminated multi-dose vials. Our molecular epidemiological investigation of hepatitis C genotype 1a, rare in Korea, will aid investigations of infection sources during future pathogen outbreaks.

Topics: 5' Untranslated Regions; Adolescent; Adult; Aged; Child; Cross Infection; Disease Outbreaks; Female; Genotype; Hepacivirus; Hepatitis C; Humans; Immunoglobulin G; Male; Middle Aged; Needle Sharing; Phylogeny; Republic of Korea; RNA, Viral; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Load; Viral Nonstructural Proteins; Young Adult

Hepatitis C virus (HCV) vaccine efforts are hampered by the extensive genetic diversity of HCV envelope glycoproteins E1 and E2. Structures of broadly neutralizing antibodies (bNAbs) (e.g., HEPC3, HEPC74) isolated from individuals who spontaneously cleared HCV infection facilitate immunogen design to elicit antibodies against multiple HCV variants. However, challenges in expressing HCV glycoproteins previously limited bNAb-HCV structures to complexes with truncated E2 cores. Here we describe crystal structures of full-length E2 ectodomain complexes with HEPC3 and HEPC74, revealing lock-and-key antibody-antigen interactions, E2 regions (including a target of immunogen design) that were truncated or disordered in E2 cores, and an antibody CDRH3 disulfide motif that exhibits common interactions with a conserved epitope despite different bNAb-E2 binding orientations. The structures display unusual features relevant to common genetic signatures of HCV bNAbs and demonstrate extraordinary plasticity in antibody-antigen interactions. In addition, E2 variants that bind HEPC3/HEPC74-like germline precursors may represent candidate vaccine immunogens.

Topics: Antibodies, Neutralizing; Antibodies, Viral; Binding Sites; Disulfides; Drug Design; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoglobulin G; Models, Molecular; Protein Conformation; Sequence Alignment; Viral Envelope Proteins; Viral Hepatitis Vaccines; X-Ray Diffraction

The role that broadly neutralizing antibodies (bNAbs) play in natural clearance of human hepatitis C virus (HCV) infection and the underlying mechanisms remain unknown. Here, we investigate the mechanism by which bNAbs, isolated from two humans who spontaneously cleared HCV infection, contribute to HCV control. Using viral gene sequences amplified from longitudinal plasma of the two subjects, we found that these bNAbs, which target the front layer of the HCV envelope protein E2, neutralized most autologous HCV strains. Acquisition of resistance to bNAbs by some autologous strains was accompanied by progressive loss of E2 protein function, and temporally associated with HCV clearance. These data demonstrate that bNAbs can mediate clearance of human HCV infection by neutralizing infecting strains and driving escaped viruses to an unfit state. These immunopathologic events distinguish HCV from HIV-1 and suggest that development of an HCV vaccine may be achievable.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antibody Specificity; Base Sequence; Binding Sites; Cell Line; Cricetulus; Epitopes; Female; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; HIV-1; Humans; Immunologic Memory; Male; Models, Molecular; Mutagenesis, Site-Directed; Viral Envelope Proteins; Viral Load

Elicitation of neutralizing antibody responses against hepatitis C virus (HCV) has been a challenging goal. While the E2 subunit of the HCV envelope glycoprotein complex is a promising target for generating cross-genotype neutralizing antibodies, vaccinations with soluble E2 immunogens generally induce weak neutralizing antibody responses. Here, E2 immunogens (i.e., E2.661 and E2c.661) were loaded into lipid-based nanovaccines and examined for induction of neutralizing antibody responses. Compared with soluble E2 immunogens, E2 nanoparticles elicited 6- to 20-fold higher E2-specific serum IgG titers in mice. Importantly, E2 vaccine nanoparticles analyzed at a single particle level with a flow cytometry-based method revealed interesting dynamics between epitope display on the surfaces of nanoparticles in vitro and induction of neutralizing antibody responses in vivo. E2c.661 nanoparticles that are preferentially bound by a broadly neutralizing antibody, HCV1, in vitro elicit neutralizing antibody responses against both autologous and heterologous HCV virions in vivo. In stark contrast, E2.661 nanoparticles with reduced HCV1-antibody binding in vitro mainly induce autologous neutralizing antibody responses in vivo. These results show that rationale antigen design coupled with interrogation of epitope display on vaccine nanoparticles at a single particle level may aid in vaccine development toward achieving neutralizing antibody responses in vivo.

Topics: Animals; Antibodies, Neutralizing; Antibody Formation; Drug Carriers; Hepacivirus; Hepatitis C; Humans; Immunoglobulin G; Mice; Nanoparticles; Viral Envelope Proteins; Viral Hepatitis Vaccines

There are numerous coding and non-coding variants in the SCARB1 gene that encodes scavenger receptor class B member 1 (SR-BI), a key receptor for both high density lipoproteins and hepatitis C virus (HCV). Many have been linked to clinical phenotypes, yet their impact on the HCV replication cycle is incompletely understood. The aim of this study was to analyze the impact of these variants on the molecular biology and clinical course of HCV.. We analyzed key coding non-synonymous as well as non-coding SCARB1 variants using virological in vitro and human genetics approaches.. Non-synonymous variants: S112F and T175A have greatly reduced HCV receptor function. When present on the cell surface, these variants are impaired in their ability to interact with HCV E2. Non-coding variants: The G allele in rs3782287 is associated with decreased viral load. Haplotype analysis confirmed these findings and identified haplotype rs3782287 A/rs5888 C as a risk allele associated with increased viral load. We also detected a trend towards lower hepatic SR-BI expression in individuals with the rs3782287 GG genotype associated with low viral load suggesting a potential underlying mechanism.. Coding and non-coding genetic SCARB1 variants modulate the HCV replication cycle and possibly clinical features of hepatitis C. These findings underscore the relevance of SR-BI as an HCV receptor and contribute to our understanding of inter-individual variation in HCV infection.. The cell surface receptor SR-BI (scavenger receptor class B member 1), is essential for hepatitis C virus (HCV) entry into hepatocytes. Variations in the gene coding this receptor influence infectivity and viral load. We analyzed these variations to gain a better understanding of inter-individual differences over the course of HCV infection.

Topics: Cell Line; Genetic Variation; Hepacivirus; Hepatitis C; Host-Pathogen Interactions; Humans; Polymorphism, Single Nucleotide; Scavenger Receptors, Class B; Viral Envelope Proteins; Viral Load; Virus Internalization; Virus Replication

The hepatitis C virus (HCV) glycoprotein E2 is the major target of neutralizing antibodies and is therefore highly relevant for vaccine design. Its structure features a central immunoglobulin (Ig)-like β-sandwich that contributes to the binding site for the cellular receptor CD81. We show that a synthetic peptide corresponding to a β-strand of this Ig-like domain forms an α-helix in complex with the anti-E2 antibody DAO5, demonstrating an inside-out flip of hydrophobic residues and a secondary structure change in the composite CD81 binding site. A detailed interaction analysis of DAO5 and cross-competing neutralizing antibodies with soluble E2 revealed that the Ig-like domain is trapped by different antibodies in at least two distinct conformations. DAO5 specifically captures retrovirus particles bearing HCV glycoproteins (HCVpp) and infectious cell culture-derived HCV particles (HCVcc). Infection of cells by DAO5-captured HCVpp can be blocked by a cross-competing neutralizing antibody, indicating that a single virus particle simultaneously displays E2 molecules in more than one conformation on its surface. Such conformational plasticity of the HCV E2 receptor binding site has important implications for immunogen design.

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Antibodies, Viral; Binding Sites; Crystallography, X-Ray; Epitopes; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Immunoglobulin Domains; Protein Binding; Protein Conformation; Tetraspanin 28; Viral Envelope Proteins; Viral Hepatitis Vaccines; Virus Internalization

The roles of CD81 in the hepatitis C virus (HCV) life cycle are multiple but remain ill characterized. CD81 is known to interact with the HCV glycoproteins as an attachment factor. It also has an important role in the post-attachment entry process. Its interaction with claudin-1, for example, is vital for viral uptake and trafficking. Furthermore, CD81 and its role in membrane organization and trafficking are thought to play a pivotal role in HCV replication. Some of these functions are particularly limited to human CD81; others can be substituted with CD81 molecules from other species. However, with the exception of the large extracellular loop sequence, the structure-function analysis of CD81 in the HCV infectious cycle remains ill characterized. We describe here the fusion molecules between the large extracellular loops of human or mouse CD81 and lipid-raft-associated or unassociated GPI anchors. These fusion molecules have strong antiviral activity in a dominant negative fashion, independent of membrane raft association. Their expression in the hepatoma cell line Huh7.5 blocks HCV uptake, transmission and replication. These molecules will be useful to decipher the various roles of CD81 in the HCV life cycle and transmission in more detail.

Topics: Animals; Cell Line, Tumor; HEK293 Cells; HeLa Cells; Hepacivirus; Hepatitis C; HIV-1; Humans; Membrane Microdomains; Mice; Protein Binding; Tetraspanin 28; Viral Envelope Proteins; Virus Attachment; Virus Internalization; Virus Replication

To investigate the effect of a single amino acid mutation in human class B scavenger receptor I (SR-BI) on the infectivity of cell culture-derived hepatitis C virus (HCVcc) in SR-BI knock-down Huh7-siSR-BI cells.. Site-directed mutagenesis was used to construct the SR-BI S112F mutation, and the mutation was confirmed by nucleotide sequencing. SR-BI knock-down Huh7-siSR-BI cells were transfected with SR-BI S112F, SR-BI wild type (WT) and control plasmids, and then infected with HCVpp (HCV pseudoparticles) and hepatitis C virus derived from cell culture (HCVcc). A fluorescence assay was performed to analyze the effect of the S112F mutation on HCV entry; quantitative real-time PCR, immunofluorescence, and Western blot assays were used to analyze the effect of the S112F mutation on HCV infectivity. CHO cells expressing WT and SR-BI S112F were incubated with the HCV E2 protein expressed in HEK 293T cells, and flow cytometry was performed to examine the ability of SR-BI S112F to bind to the HCV E2 protein. Huh7-siSR-BI cells were transfected with SR-BI WT and the S112F mutant, and then DiI-HDL was added and images captured under the microscope to assess the ability of SR-BI S112F to take up HDL.. The SR-BI S112F mutation was successfully constructed. The S112F mutation decreased the expression of the SR-BI mRNA and protein. SR-BI S112F decreased HCV entry and HCVcc infectivity in Huh7-siSR-BI cells. The S112F mutation impaired the binding of SR-BI to HCV E2 protein and decreased the HDL uptake of SR-BI.. The S112F single amino acid mutation in SR-BI decreased the levels of the SR-BI mRNA and protein, as well as the ability of SR-BI to bind to the HCV E2 protein. Amino acid 112 in SR-BI plays important roles in HCV entry and the infectivity of HCVcc

Topics: Animals; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Down-Regulation; Flow Cytometry; Gene Knockdown Techniques; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Mutagenesis, Site-Directed; Point Mutation; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Scavenger Receptors, Class B; Transfection; Viral Envelope Proteins; Virus Internalization

The existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines.. First, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments.. Our binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism).. These results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development.

Topics: Aedes; Animals; Cell Line; Genotype; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Insect Vectors; Mutation; Peptides; Phylogeny; Polymerase Chain Reaction; Polymorphism, Genetic; RNA, Viral; Sequence Analysis; Viral Envelope Proteins; Virus Replication

Topics: Adult; Bayes Theorem; Coinfection; Computational Biology; Female; Genetic Variation; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; High-Throughput Nucleotide Sequencing; HIV Infections; HIV Seropositivity; HIV-1; Humans; Immunity, Humoral; Infant; Infectious Disease Transmission, Vertical; Pregnancy; Pregnancy Complications, Infectious; Quasispecies; Risk Factors; Viral Envelope Proteins

Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP). HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus.

Topics: Amino Acid Sequence; Cell Line; HEK293 Cells; Hepacivirus; Hepatitis C; Host-Pathogen Interactions; Humans; Models, Biological; Mutation; Protein Processing, Post-Translational; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins; Virulence; Virus Assembly

A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography.. A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antigens, Viral; Bacterial Proteins; Chromatography, Agarose; Cross Reactions; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immune Sera; Immunoglobulin Fc Fragments; Mice; Neutralization Tests; Protein Folding; Protein Multimerization; Recombinant Fusion Proteins; Vaccination; Vaccines, Synthetic; Viral Envelope Proteins; Viral Hepatitis Vaccines

A vaccine that prevents hepatitis C virus (HCV) infection is urgently needed to support an emerging global elimination program. However, vaccine development has been confounded because of HCV's high degree of antigenic variability and the preferential induction of type-specific immune responses with limited potency against heterologous viral strains and genotypes. We showed previously that deletion of the three variable regions from the E2 receptor-binding domain (Δ123) increases the ability of human broadly neutralizing antibodies (bNAbs) to inhibit E2-CD81 receptor interactions, suggesting improved bNAb epitope exposure. In this study, the immunogenicity of Δ123 was examined. We show that high-molecular-weight forms of Δ123 elicit distinct antibody specificities with potent and broad neutralizing activity against all seven HCV genotypes. Antibody competition studies revealed that immune sera raised to high-molecular-weight Δ123 was poly specific, given that it inhibited the binding of human bNAbs directed to three major neutralization epitopes on E2. By contrast, the immune sera raised to monomeric Δ123 predominantly blocked the binding of a non-neutralizing antibody to Δ123, while having reduced ability to block bNAb binding to E2, and neutralization was largely toward the homologous genotype. This increased ability of oligomeric Δ123 to generate bNAbs correlates with occlusion of the non-neutralizing face of E2 in this glycoprotein form.. The results from this study reveal new information on the antigenic and immunogenic potential of E2-based immunogens and provide a pathway for the development of a simple, recombinant protein-based prophylactic vaccine for HCV with potential for universal protection. (Hepatology 2017;65:1117-1131).

Topics: Animals; Antibodies, Neutralizing; Antibody Specificity; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Epitopes; Genotype; Guinea Pigs; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Random Allocation; Statistics, Nonparametric; Viral Envelope Proteins; Viral Hepatitis Vaccines

Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3-4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare cross-genotype conserved epitope. By analyzing the genotype 1a envelope proteins (E1/E2) of recovered Core-NS2 recombinant H77/JFH1ΔHVR1 and performing reverse genetic studies we found that resistance to AR5A was caused by substitution L665W, also conferring resistance to the parental H77/JFH1. The mutation did not induce viral fitness loss, but abrogated AR5A binding to HCV particles and intracellular E1/E2 complexes. Culturing J6/JFH1ΔHVR1 (genotype 2a), for which fitness was decreased by L665W, with AR5A generated AR5A-resistant viruses with the substitutions I345V, L665S, and S680T, which we introduced into J6/JFH1 and J6/JFH1ΔHVR1. I345V increased fitness but had no effect on AR5A resistance. L665S impaired fitness and decreased AR5A sensitivity, while S680T combined with L665S compensated for fitness loss and decreased AR5A sensitivity even further. Interestingly, S680T alone had no fitness effect but sensitized the virus to AR5A. Of note, H77/JFH1L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2-6 parental and HVR1-deleted variants (not available for genotype 4a) we observed diverse effects on viral fitness and a universally pronounced reduction in AR5A sensitivity. Thus, we were able to take advantage of the neutralization-sensitive HVR1-deleted viruses to rapidly generate escape viruses aiding our understanding of the divergent escape pathways used by HCV to evade AR5A.

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Antibodies, Viral; Cell Line; Epitopes, B-Lymphocyte; Hepacivirus; Hepatitis C; Humans; Immune Evasion; Immunoprecipitation; Mutation; Viral Envelope Proteins; Viral Proteins

Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes.

Topics: Algorithms; Amino Acid Sequence; Antibodies, Neutralizing; Enzyme-Linked Immunosorbent Assay; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; High-Throughput Nucleotide Sequencing; Humans; Immune Evasion; Mutagenesis, Site-Directed; Neutralization Tests; Phylogeny; Polymorphism, Genetic; Scavenger Receptors, Class B; Viral Envelope Proteins

The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2).. Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc).. In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes.. Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool.

Topics: Antibodies, Monoclonal, Humanized; Cell Engineering; Cells, Cultured; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Immunotherapy; Receptors, Antigen, T-Cell; T-Lymphocytes; Viral Envelope Proteins

Hepatitis C virus (HCV) envelope E2 is a glycoprotein that are implicated in HCV infection by facilitating its entry and immune evasion, which in turn leads to hepatitis, hepatocellular carcinoma and other chronic liver diseases. It is reported that the HCV E2 stem region comprise a functional region for HCV entry; however, the roles and underlying mechanism of these conserved residues on the E2 protein in the immune response after HCV infection are still not well defined. In this study, we synthesized 30 aa peptides containing residues 705-734 (E2-705) of HCV E2 using the solid-phase peptide synthesis (SPPS) method. The characteristics of the synthesized peptides were identified by Western blot and cell culture derived HCV particles (HCVcc) infection blocking assay. ELISA and flow cytometry assays were employed to determine the effect of the synthesized peptide on dendritic cells (DCs) response and CD4(+) T cell activation. Results showed that the synthesized E2-705 peptides binds to DCs by interaction with DC-SIGN receptor. E2-705 peptides induced the maturation of infected DCs to a similar extent with recombinant HCV E2 as reflected by the antigen uptake potential and allostimulatory capacity. Furthermore, the E2-705 peptides increased the production of IL-12, CD80 and CD86 but reduced the IL-10 in DCs, in which p38 MAPK signaling might be involved. These results suggest that the carboxyl-terminus beyond the core ectodomain of HCV E2 protein may play a key role in immunoreaction of HCV infection, giving a new understanding of HCV E2 and a novel target for the design of HCV vaccines or inhibitors.

Topics: CD4-Positive T-Lymphocytes; Cell Adhesion Molecules; Cell Differentiation; Cells, Cultured; Dendritic Cells; Hepacivirus; Hepatitis C; Humans; Interleukin-10; Interleukin-12; Lectins, C-Type; Lymphocyte Activation; MAP Kinase Signaling System; Peptide Fragments; Receptors, Cell Surface; Signal Transduction; Viral Envelope Proteins; Viral Hepatitis Vaccines

Injection drug users uninfected by hepatitis C virus (HCV) despite likely repeated exposure through high-risk behaviour are well documented. Factors preventing infection in these individuals are incompletely understood. Here, we looked for anti-HCV-envelope antibody responses in a cohort of repeatedly exposed but uninfected subjects. Forty-two hepatitis C diagnostic antibody- and RNA-negative injection drug users at high risk of exposure were studied and findings compared to healthy controls and cases with chronic HCV infection. Purified IgGs from sera were tested by ELISA for binding to genotype 1a and 3a envelope glycoproteins E1E2 with further testing for IgG and IgM reactivity against soluble E2. Virus-neutralizing activity was assessed using an HCV pseudoparticle system. Uninfected subjects demonstrated significantly greater IgG and IgM reactivities to envelope glycoproteins than healthy controls with IgG from 6 individuals additionally showing significant neutralization. This study is the first to describe humoral immunological responses targeting the HCV envelope, important for viral neutralization, in exposed uninfected individuals. A subset of these cases also had evidence of viral neutralization via anti-envelope antibodies. In addition to confirming viral exposure, the presence of specific anti-envelope antibodies may be a factor that helps these individuals resist HCV infection.

Topics: Adult; Antibody Formation; Disease Resistance; Drug Users; Environmental Exposure; Enzyme-Linked Immunosorbent Assay; Female; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Middle Aged; Neutralization Tests; Substance Abuse, Intravenous; Viral Envelope Proteins

Hepatitis C virus (HCV) represents a major global health threat. The envelope glycoproteins, E1-E2 of HCV play an important role in infection by binding to hepatocyte surface receptors leading to viral entry. Several regions on the E1-E2 are conserved for maintaining structural stability, despite the high mutation rate of HCV. Identification of antigenic determinants in these domains would aid in the development of anti-virals. The present study was aimed to delineate neutralizing epitopes by generating monoclonal antibodies (mAbs) to envelope proteins that can block virus binding and entry. Using HCV-like particles (HCV-LPs) corresponding to genotype 3a (prevalent in India), we obtained three mAbs specific for the E2 protein that significantly inhibited virus binding to hepatoma cells. Using overlapping protein fragments and peptides of the E2 protein, the epitopes corresponding to the mAbs were delineated. MAbs H6D3 and A10F2 recognise sequential linear epitopes, whereas, mAb E3D8 recognises a discontinuous epitope. The epitope of mAb E3D8 overlaps with the CD81 receptor-binding site and that of mAb A10F2 with the hypervariable region 2 of the E2 protein. The epitopes corresponding to these mAbs are distinct and unique. A combination of these antibodies significantly inhibited HCV binding and entry in both HCV pseudoparticle (in vitro) and HCV cell culture (ex vivo) system compared to the mAbs alone (P<0.0001). In conclusion, our findings support the potential of employing a cocktail of neutralizing mAbs in the management of HCV infection.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Cell Line, Tumor; CHO Cells; Cricetulus; Genotype; Hepacivirus; Hepatitis C; Humans; Immunodominant Epitopes; India; Mice; Protein Binding; Tetraspanin 28; Viral Envelope Proteins; Virion; Virus Attachment; Virus Internalization

While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties.. Characterizing variant viruses can reveal new information about the life cycle of HCV and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth-enhancing mutations reduced the dependence on a key entry receptor, SR-BI. By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof of principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability.

Topics: Adaptation, Physiological; Amino Acid Substitution; Antibodies, Viral; Cell Line; CRISPR-Cas Systems; Gene Library; Genetic Engineering; Genome, Viral; Hepacivirus; Hepatitis C; Host-Pathogen Interactions; Humans; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Scavenger Receptors, Class B; Tetraspanin 28; Viral Envelope Proteins

Acute hepatitis C virus (HCV) infection culminates in viral persistence in the majority of cases. Abs that recognize the envelope glycoproteins E1 and E2 are generated during the late stages of acute infection, yet their contribution to spontaneous viral clearance remains controversial. Investigation of the humoral responses during acute HCV infection have been limited by the inability to directly identify and characterize HCV-specific B cells. In this study we describe the development of a novel tetramer of the E2 glycoprotein ectodomain (J6, genotype 2a strain), which allowed us to visualize E2-specific B cells longitudinally in the peripheral blood of HCV-infected individuals. HCV-specific class-switched memory B cells were detected in 3 out of 7 participants during late acute infection, with a mean frequency of 0.63% for positive samples (range 0.16-0.67%) and in 7 out of 7 participants with chronic infection with a mean frequency of 0.47% (range 0.20-0.78%). In a cross-sectional study, E2 tetramer positive population was detected in 28 out of 31 chronically infected individuals. Deep sequencing of the BCR from E2-specific class-switched memory B cells sorted from two independent participants revealed a focused repertoire suggestive of clonal selection. Tetramer-specific B cells exhibited skewed CDR3 length distribution and increased mutation frequency compared with naive B cells. This BCR profile is indicative of clonal expansion and affinity maturation. E2 tetramer allows for specific and sensitive ex vivo characterization of rare HCV-specific B cells in infected individuals, and will enable researchers to gain a better understanding of humoral immunity in HCV infection.

Topics: Adult; B-Lymphocytes; Cross-Sectional Studies; Female; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Immunity, Humoral; Immunologic Memory; Immunologic Tests; Male; Protein Binding; Protein Engineering; Viral Envelope Proteins

The present work aimed at 1) characterization of the E1 and E2 proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and antibodies raised using mammalian expression constructs encoding the protein. The region encoding the E1 and E2 proteins was amplified by RT-PCR from RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-PROTEIN server and PSI-PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by nucleotide and protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The B-cell epitope CFTPSPVVV at position 203 and the T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope proteins using mouse antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed protein. The antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating antigens in patients' sera with good sensitivity that correlated with liver enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = -0.5439, P = 0.0019 for AST). Moreover, combining the HCV-E-encoding construct with extracts prepared from Echinacea purpurea and Nigella sativa prior to immunizing mice significantly (P < 0.05) increased both the humoral (14.9- to 20-fold increase in antibodies) and the cellular (CD4(+) and cytotoxic CD8(+)- T lymphocytes) responses compared to mice that received the DNA construct alone or PBS-treated mice. Both recombinant HCV-E protein preparations and antibodies raised using the HCV-E-encoding mammalian expr

Topics: Animals; Echinacea; Egypt; Epitopes, B-Lymphocyte; Epitopes, T-Lymphocyte; Female; Gene Expression; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Mice; Mice, Inbred BALB C; Models, Molecular; Molecular Sequence Data; Nigella sativa; Phylogeny; Sequence Alignment; Viral Envelope Proteins

To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies.

Topics: Animals; Cell Line; Cricetinae; Green Fluorescent Proteins; Hepacivirus; Hepatitis C; Humans; Models, Biological; Vesicular Stomatitis; Viral Envelope Proteins

Chronic hepatitis C virus (HCV) infection predisposes patients to develop liver failure after acetaminophen (APAP) overdose. Mechanisms involved in this were explored using transgenic mice expressing the HCV structural proteins core, E1 and E2. Treatment of C57BL/6J mice with 200 mg/kg body weight APAP resulted in significant liver injury at 6 h as indicated by elevated ALT levels, focal centrilobular necrosis and nuclear DNA fragmentation. HCV transgenic mice showed a variable response, with approximately half the animals showing exacerbation of all parameters of liver injury, while the other half was protected. HCV transgenic mice with higher liver injury had lower liver glutathione levels, elevated mitochondrial oxidative stress and enhanced release of apoptosis-inducing factor (AIF) from the mitochondria. This was accompanied by induction of a higher ER stress response and induction of autophagy. Transgenic animals showing protection against liver injury had a robust recovery of liver glutathione content at 6 h when compared to wild-type animals, accompanied by reduction in mitochondrial oxidative stress and AIF release. This was accompanied by an elevation in glutathione S-transferase mRNA levels and activity, which suggests that an efficient clearance of the reactive intermediate may contribute to the protection against APAP hepatotoxicity in these mice. These results demonstrate that while HCV infection could exacerbate APAP-induced liver injury due to induction and amplification of mitochondrial oxidant stress, it could also protect against injury by activation of APAP scavenging mechanisms.

Topics: Acetaminophen; Animals; Autophagy; Chemical and Drug Induced Liver Injury; Hepatitis C; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria, Liver; Oxidative Stress; Viral Envelope Proteins

Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection.. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA).. The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the 30 sera of known genotypes. The antigens did not detect antibodies to genotype-3a, but detected antibodies to all genotypes and did not discriminate them genotype wise. A panel of 175 of HCV-suspected serum samples was subjected to comparative analysis with our in-house ELISA assay and with commercial HCV screening assays. After subjecting the results to the formulas for determining the quality parameters, immunoblot assay had 100% sensitivity and specificity, while the ELISA assay had 100% sensitivity and 98.8% specificity as compared to commercially available assays.. This study indicates that a mixture of Core and E2 antigens are potentially valuable antigens and there is the possibility of developing serological assays for monitoring HCV infection.

Topics: Cross Reactions; Early Diagnosis; Escherichia coli; Genetic Vectors; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Immunoblotting; Recombinant Proteins; Sensitivity and Specificity; Viral Core Proteins; Viral Envelope Proteins

Perinatal exposure to hepatitis C virus (HCV) antigens during pregnancy may affect the developing immune system in the fetus. We aimed to study the perinatal transmission of HCV structural and non-structural antigens.. Sera from 402 pregnant mothers were tested for anti-HCV antibody and HCV RNA. HCV antigens were determined in sera from 101 HCV-infected mothers and their cord blood.. In both serum and cord blood samples, HCV NS4 (non-structural 4) at 27 kDa, E1 (envelope 1) at 38 kDa and E2 (envelope 2) at 40 kDa were identified, purified and quantified using western blotting, electroelution and ELISA. Maternal sera and neonate cord blood samples had similar detection rates for NS4 (94.1%), E1 (90.1%) and E2 (90.1%). The mean maternal serum levels (optical density, OD) of HCV NS4 (0.87 ± 0.01), E1 (0.86 ± 0.01) and E2 (0.85 ± 0.01) did not differ significantly (p > 0.05) from those of neonatal cord blood (0.83 ± 0.01, 0.87 ± 0.01 and 0.85 ± 0.01, respectively). Also, strong correlations (p < 0.0001) were shown between sera and cord blood sample levels of HCV NS4, r = 0.77; E1, r = 0.76 and E2, r = 0.80. The vertical transmission of these antigens in vaginal delivery did not differ significantly (p > 0.05) from those in caesarean section.. These findings indicate that vertical transmission of HCV NS4, E1 and E2 antigens was very high. Thus, exposure to these antigens may influence the developing immune responses to natural infection or future vaccination.

Topics: Adult; Blotting, Western; Female; Fetal Blood; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Infant, Newborn; Infectious Disease Transmission, Vertical; Pregnancy; Pregnancy Complications, Infectious; Viral Envelope Proteins; Viral Nonstructural Proteins

The E2 glycoprotein of Hepatitis C virus (HCV) is a major target of the neutralizing antibody (NAb) response with the majority of epitopes located within its receptor binding domain (RBD; 384-661). Within E2 are three variable regions located at the N-terminus (HVR1; 384-411), and internally at 460-480 (HVR2) and 570-580 [intergenotypic variable region (igVR)], all of which lie outside a conserved core domain that contains the CD81 binding site, essential for attachment of virions to host cells and a major target of NAbs. In this study, we examined the evolution of the E1 and E2 region in two patients infected with genotype 3a virus. Whereas one patient was able to clear the acute infection, the other developed a chronic infection. Mutations accumulated at multiple positions within the N-terminal HVR1 as well as within the igVR in both patients over time, whereas mutations in HVR2 were observed only in the chronically infected patient. Mutations within or adjacent to the CD81 contact site were observed in both patients but were less frequent and more conservative in the patient that cleared his/her infection. The evolution of CD81 binding function and antigenicity was examined with longitudinal E2 RBD sequences. The ability of the RBD to bind CD81 was completely lost by week 108 in the patient that developed chronic HCV. In the second patient, the ability of the week 36 RBD, just prior to viral clearance, to bind CD81 was reduced ~50% relative to RBD sequences obtained earlier. The binding of a NAb specific to a conserved epitope located within E2 residues 411-428 was significantly reduced by week 108 despite complete conservation of its epitope suggesting that E2 antigenicity is allosterically modulated. The exposure of non-neutralizing antibody epitopes was similarly explored and we observed that the epitope of 3 out of 4 non-NAbs were significantly more exposed in the RBDs representing the late timepoints in the chronic patient. By contrast, the exposure of non-neutralizing epitopes was reduced in the patient that cleared his/her infection and could in part be attributed to sequence changes in the igVR. These studies reveal that during HCV infection, the exposure of the CD81 binding site on E2 becomes increasingly occluded, and the antigenicity of the E2 RBD towards both neutralizing and non-neutralizing antibodies is modulated via allosteric mechanisms.

Topics: Allosteric Regulation; Amino Acid Sequence; Antibodies, Neutralizing; Antibodies, Viral; Evolution, Molecular; Genotype; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Mutation, Missense; Protein Binding; Protein Interaction Domains and Motifs; Tetraspanin 28; Viral Envelope Proteins

Pegylated interferon and ribavirin combination therapy effectively suppresses viral replication in 50 %-60 % of hepatitis C virus (HCV)-infected patients. However, HCV-infected patients often display varied responses to therapy, and strains of subtype lb (the most widespread HCV subtype worldwide) have more-severe clinical manifestations, greater viral loads, and poorer responses to interferon treatment. Therefore, understanding the genomic variability of HCV is crucial to treatment of HCV infection. In this study, we used the appropriate software to analyze the nucleotide, and amino acid sequences of the envelope proteins (E1 and E2) of HCV to investigate the extent of their variability in several HCV subtypes (1a, 1b, 2a, 2b, 3a, 4a, 5a and 6a) and calculated the ratio of nonsynonymous to synonymous substitutions (dN/dS) in these proteins to investigate the immunological pressure acting on them. We also predicted the N-glycosylation sites in E1 and E2 to determine their association with viral neutralization. We found that E1 is more variable, has a higher dN/dS ratio, and has more N-glycosylation sites than E2 in HCV subtype 1b. This indicates that the variability of E1, its dN/dS ratio, and its degree of N-glycosylation might play an important role in the treatment of infection with HCV subtype 1b.

Topics: Genetic Variation; Genotype; Glycosylation; Hepacivirus; Hepatitis C; Humans; Viral Envelope Proteins

Lipoprotein components are crucial factors for hepatitis C virus (HCV) assembly and entry. As hepatoma cells producing cell culture-derived HCV (HCVcc) particles are impaired in some aspects of lipoprotein metabolism, it is of upmost interest to biochemically and functionally characterize the in vivo produced viral particles, particularly regarding how lipoprotein components modulate HCV entry by lipid transfer receptors such as scavenger receptor BI (SR-BI). Sera from HCVcc-infected liver humanized FRG mice were separated by density gradients. Viral subpopulations, termed HCVfrg particles, were characterized for their physical properties, apolipoprotein association, and infectivity. We demonstrate that, in contrast to the widely spread distribution of apolipoproteins across the different HCVcc subpopulations, the most infectious HCVfrg particles are highly enriched in apoE, suggesting that such apolipoprotein enrichment plays a role for entry of in vivo derived infectious particles likely via usage of apolipoprotein receptors. Consistent with this salient feature, we further reveal previously undefined functionalities of SR-BI in promoting entry of in vivo produced HCV. First, unlike HCVcc, SR-BI is a particularly limiting factor for entry of HCVfrg subpopulations of very low density. Second, HCVfrg entry involves SR-BI lipid transfer activity but not its capacity to bind to the viral glycoprotein E2. In conclusion, we demonstrate that composition and biophysical properties of the different subpopulations of in vivo produced HCVfrg particles modulate their levels of infectivity and receptor usage, hereby featuring divergences with in vitro produced HCVcc particles and highlighting the powerfulness of this in vivo model for the functional study of the interplay between HCV and liver components.

Topics: Animals; Apolipoproteins E; Cell Line; Disease Models, Animal; Hepacivirus; Hepatitis C; Humans; Liver; Mice; Mice, Knockout; Scavenger Receptors, Class B; Viral Envelope Proteins; Virus Internalization

High rates of hepatitis C virus (HCV) reinfections among HIV-infected men who have sex with men (MSM) following clearance of a primary infection suggest absence of protective immunity. Here, we investigated the incidence of HCV super and reinfections in 85 HIV-infected MSM with incident HCV infection.. Serial sequencing of a fragment of NS5B and the HCV envelope was used to longitudinally characterize the virus. If the primary genotype was still present at the most recent viremic time point, as indicated by the NS5B sequence analysis, serial envelope 2/hypervariable region 1 (E2/HRV1) sequence analysis was performed to distinguish a new infection with the same genotype (clade switch) from intrahost evolution. Incidence rate and cumulative incidence of secondary infections were estimated, and the effect of the primary genotype (1a versus non1) on the risk of acquiring a second infection with the same genotype was determined using Cox proportional-hazards analysis.. Among 85 patients with a median follow-up of 4.8 years, incidence rate of secondary infections was 5.39 cases/100 person-years (95% confidence interval 3.34-8.26). Cumulative incidence of genotype switches was markedly higher than the cumulative incidence of clade switches (26.7 versus 4.8% at 5 years, respectively). In patients with HCV-1a as primary infection, the risk for acquiring another HCV-1a infection was reduced compared to those with a primary non-HCV-1a subsequently acquiring HCV-1a (hazard ratio 0.25, 95% confidence interval 0.07-0.93).. Risk of acquiring a secondary infection with the primary genotype was strikingly reduced compared with the risk of acquiring a secondary infection with a different genotype.

Topics: Adult; Genotype; Hepacivirus; Hepatitis C; HIV Infections; Homosexuality, Male; Humans; Incidence; Male; Middle Aged; Molecular Epidemiology; Prospective Studies; Retrospective Studies; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Nonstructural Proteins

Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1-6 was determined. We defined a domain comprising amino acids (aa) 192-221, 232-251, 262-281 and 292-331 of E1, and 421-543, 564-583, 594-618 and 634-673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444-463) and PUHI45 (aa 604-618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Cell Line; Epitopes; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Mice; Peptide Library; Species Specificity; Viral Envelope Proteins; Viral Hepatitis Vaccines

T cell receptor (TCR) signaling is required for T-cell activation, proliferation, differentiation, and effector function. Hepatitis C virus (HCV) infection is associated with impaired T-cell function leading to persistent viremia, delayed and inconsistent antibody responses, and mild immune dysfunction. Although multiple factors appear to contribute to T-cell dysfunction, a role for HCV particles in this process has not been identified. Here, we show that incubation of primary human CD4+ and CD8+ T-cells with HCV RNA-containing serum, HCV-RNA containing extracellular vesicles (EVs), cell culture derived HCV particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibited TCR-mediated signaling. Since HCVpp's contain only E1 and E2, we examined the effect of HCV E2 on TCR signaling pathways. HCV E2 expression recapitulated HCV particle-induced TCR inhibition. A highly conserved, 51 nucleotide (nt) RNA sequence was sufficient to inhibit TCR signaling. Cells expressing the HCV E2 coding RNA contained a short, virus-derived RNA predicted to be a Dicer substrate, which targeted a phosphatase involved in Src-kinase signaling (PTPRE). T-cells and hepatocytes containing HCV E2 RNA had reduced PTPRE protein levels. Mutation of 6 nts abolished the predicted Dicer interactions and restored PTPRE expression and proximal TCR signaling. HCV RNA did not inhibit distal TCR signaling induced by PMA and Ionomycin; however, HCV E2 protein inhibited distal TCR signaling. This inhibition required lymphocyte-specific tyrosine kinase (Lck). Lck phosphorylated HCV E2 at a conserved tyrosine (Y613), and phospho-E2 inhibited nuclear translocation of NFAT. Mutation of Y613 restored distal TCR signaling, even in the context of HCVpps. Thus, HCV particles delivered viral RNA and E2 protein to T-cells, and these inhibited proximal and distal TCR signaling respectively. These effects of HCV particles likely aid in establishing infection and contribute to viral persistence.

Topics: Base Sequence; Conserved Sequence; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Hepacivirus; Hepatitis C; Humans; Immunoblotting; Immunoprecipitation; Lymphocyte Activation; Polymerase Chain Reaction; Receptors, Antigen, T-Cell; RNA, Viral; T-Lymphocytes; Viral Envelope Proteins; Virion

Hepatitis C virus (HCV) E2 protein binds to CD81, which is a component of the B cell co-stimulatory complex. The E2-CD81 interaction leads to B cell proliferation, protein tyrosine phosphorylation and to the hypermutation of immunoglobulin genes. Epidemiological studies have reported a high prevalence of B cell non-Hodgkin lymphoma (NHL) in HCV-positive patients, suggesting a potential association between HCV and Epstein-Barr virus (EBV) in the genesis of B lymphocyte proliferative disorders. In the present study, in order to investigate the association between EBV and HCV in B cells, we created an in vitro EBV-induced B cell transformation model. CD81 was gradually overexpressed during transformation by EBV. B cells isolated from HCV-positive patients grew more rapidly and clumped together earlier than B cells isolated from healthy donors following EBV infection. Pre-stimulation of CD81 expressed by resting B cells with anti-CD81 monoclonal antibody (mAb) or HCV E2 accelerated the generation of lymphoblastoid cell lines (LCLs) by EBV infection. These cells proliferated prominently through the early expression of interleukin-10 and intracellular latent membrane protein (LMP)-l. By contrast, the overexpression of CD81 on EBV-transformed B cells by anti-CD81 mAb or HCV E2 protein induced apoptosis through reactive oxygen species (ROS)-mediated mitochondrial dysfunction. These results suggest that the engagement of CD81 expressed by B cells has differential effects on B cell fate (proliferation or apoptosis) according to EBV infection and the expression level of CD81.

Topics: Adult; Antibodies; Apoptosis; Apoptosis Regulatory Proteins; B-Lymphocytes; Blotting, Western; Cell Proliferation; Cell Transformation, Viral; Cells, Cultured; Epstein-Barr Virus Infections; Female; Hepacivirus; Hepatitis C; Herpesvirus 4, Human; Host-Pathogen Interactions; Humans; Male; Membrane Potential, Mitochondrial; Microscopy, Confocal; Middle Aged; Protein Binding; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Tetraspanin 28; Viral Envelope Proteins

The human immunodeficiency virus 1 (HIV-1) epidemic in China historically stemmed from intravenous drug users (IDUs) in Yunnan. Due to a shared transmission route, hepatitis C virus (HCV)/HIV-1 co-infection is common. Here, we investigated HCV genetic characteristics and baseline drug resistance among HIV-infected IDUs in Yunnan.. Blood samples of 432 HIV-1/HCV co-infected IDUs were collected from January to June 2014 in six prefectures of Yunnan Province. Partial E1E2 and NS5B genes were sequenced. Phylogenetic, evolutionary and genotypic drug resistance analyses were performed.. Among the 293 specimens successfully genotyped, seven subtypes were identified, including subtypes 3b (37.9%, 111/293), 3a (21.8%, 64/293), 6n (14.0%, 41/293), 1b (10.6%, 31/293), 1a (8.2%, 24/293), 6a (5.1%, 15/293) and 6u (2.4%, 7/293). The distribution of HCV subtypes was mostly related to geographic location. Subtypes 3b, 3a, and 6n were detected in all six prefectures, however, the other four subtypes were detected only in parts of the six prefectures. Phylogeographic analyses indicated that 6n, 1a and 6u originated in the western prefecture (Dehong) and spread eastward and showed genetic relatedness with those detected in Burmese. However, 6a originated in the southeast prefectures (Honghe and Wenshan) bordering Vietnam and was transmitted westward. These subtypes exhibited different evolutionary rates (between 4.35×10-4 and 2.38×10-3 substitutions site-1 year-1) and times of most recent common ancestor (tMRCA, between 1790.3 and 1994.6), suggesting that HCV was multiply introduced into Yunnan. Naturally occurring resistance-associated mutations (C316N, A421V, C445F, I482L, V494A, and V499A) to NS5B polymerase inhibitors were detected in direct-acting antivirals (DAAs)-naïve IDUs.. This work reveals the temporal-spatial distribution of HCV subtypes and baseline HCV drug resistance among HIV-infected IDUs in Yunnan. The findings enhance our understanding of the characteristics and evolution of HCV in IDUs and are valuable for developing HCV prevention and management strategies for this population.

Topics: Adult; China; Coinfection; Drug Resistance, Viral; Epidemics; Female; Genotype; Geography; Hepacivirus; Hepatitis C; HIV Infections; Host-Pathogen Interactions; Humans; Male; Molecular Sequence Data; Mutation; Phylogeny; Prevalence; Sequence Analysis, DNA; Substance Abuse, Intravenous; Viral Envelope Proteins; Viral Nonstructural Proteins

Despite significant advances in the treatment of hepatitis C virus (HCV) infection, the need to develop preventative vaccines remains. Identification of the best vaccine candidates and evaluation of their performance in preclinical and clinical development will require appropriate neutralization assays utilizing diverse HCV isolates. We aimed to generate and characterize a panel of HCV E1E2 glycoproteins suitable for subsequent use in vaccine and therapeutic antibody testing. Full-length E1E2 clones were PCR amplified from patient-derived serum samples, cloned into an expression vector, and used to generate viral pseudoparticles (HCVpp). In addition, some of these clones were used to generate cell culture infectious (HCVcc) clones. The infectivity and neutralization sensitivity of these viruses were then determined. Bioinformatic and HCVpp infectivity screening of approximately 900 E1E2 clones resulted in the assembly of a panel of 78 functional E1E2 proteins representing distinct HCV genotypes and different stages of infection. These HCV glycoproteins differed markedly in their sensitivity to neutralizing antibodies. We used this panel to predict antibody efficacy against circulating HCV strains, highlighting the likely reason why some monoclonal antibodies failed in previous clinical trials. This study provides the first objective categorization of cross-genotype patient-derived HCV E1E2 clones according to their sensitivity to antibody neutralization. It has shown that HCV isolates have clearly distinguishable neutralization-sensitive, -resistant, or -intermediate phenotypes, which are independent of genotype. The panel provides a systematic means for characterization of the neutralizing response elicited by candidate vaccines and for defining the therapeutic potential of monoclonal antibodies.. Hepatitis C virus (HCV) has a global burden of more than 170 million people, many of whom cannot attain the new, expensive, direct-acting antiviral therapies. A safe and effective vaccine that generates both T cell responses and neutralizing antibodies is required to eradicate the disease. Regions within the HCV surface glycoproteins E1 and E2 are essential for virus entry and are targets for neutralizing antibodies. Screening of vaccine candidates requires suitable panels of glycoproteins that represent the breadth of neutralization resistance. Use of a standard reference panel for vaccine studies will ensure comparability of data sets, as has become routine for HIV-1. Here, we describe a large panel of patient-derived HCV glycoproteins with an assessment of their neutralization sensitivity to defined monoclonal antibodies, which has enabled us to predict their likely efficacy in the wider HCV-infected population. The panel could also be important for future selection of additional therapeutic antibodies and for vaccine design.

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Cell Line; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Molecular Sequence Data; Neutralization Tests; Viral Envelope Proteins; Viral Hepatitis Vaccines

Hepatitis C virus (HCV) is one of the leading causes of chronic liver inflammatory disease (hepatitis), which often leads to more severe diseases, such as liver fibrosis, cirrhosis, and hepatocellular carcinoma. Liver fibrosis, in particular, is a major pathogenic consequence of HCV infection, and transforming growth factor β1 (TGF-β1) plays a key role in its pathogenesis. Several HCV proteins have been suggested to either augment or suppress the expression of TGF-β1 by HCV-infected cells. Here, we report that TGF-β1 levels are elevated in HCV-infected hepatocytes cultured in vitro and in liver tissue of HCV patients. Notably, the level of TGF-β1 in media from in vitro-cultured HCV-infected hepatocytes was high enough to activate primary hepatic stellate cells isolated from rats. This indicates that TGF-β1 secreted by HCV-infected hepatocytes is likely to play a key role in the liver fibrosis observed in HCV patients. Moreover, we showed that HCV E2 protein triggers the production of TGF-β1 by HCV-infected cells through overproduction of glucose-regulated protein 94 (GRP94).. Hepatic fibrosis is a critical step in liver cirrhosis caused by hepatitis C virus infection. It is already known that immune cells, including Kupffer cells, mediate liver fibrosis. Recently, several papers have suggested that HCV-infected hepatocytes also significantly produce TGF-β1. Here, we provide the first examination of TGF-β1 levels in the hepatocytes of HCV patients. Using an HCV culture system, we showed that HCV infection increases TGF-β1 production in hepatocytes. Furthermore, we confirmed that the amount of TGF-β1 secreted by HCV-infected hepatocytes was sufficient to activate primary hepatic stellate cells. To understand the molecular basis of TGF-β1 production in HCV-infected hepatocytes, we used HCV replicons and various stable cell lines. Finally, we elucidated that HCV E2 triggered TGF-β1 secretion via GRP94 mediated NF-κB activation. This study contributes to the understanding of liver fibrosis by HCV and suggests a new potential target (GRP94) for blocking liver cirrhosis in HCV patients.

Topics: Animals; Cells, Cultured; Culture Media, Conditioned; Hepacivirus; Hepatic Stellate Cells; Hepatitis C; Hepatocytes; Host-Pathogen Interactions; Humans; Liver Cirrhosis; Membrane Glycoproteins; Rats, Sprague-Dawley; Transforming Growth Factor beta1; Viral Envelope Proteins

Identification of novel drug targets and affordable therapeutic agents remains a high priority in the fight against chronic hepatitis C virus (HCV) infection. Here, we report that the cellular proteins prohibitin 1 (PHB1) and 2 (PHB2) are pan-genotypic HCV entry factors functioning at a post-binding step. While predominantly found in mitochondria, PHBs localize to the plasma membrane of hepatocytes through their transmembrane domains and interact with both EGFR and CRaf. Targeting PHB by rocaglamide (Roc-A), a natural product that binds PHB1 and 2, reduced cell surface PHB1 and 2, disrupted PHB-CRaf interaction, and inhibited HCV entry at low nanomolar concentrations. A structure-activity analysis of 32 synthetic Roc-A analogs indicated that the chiral, racemic version of aglaroxin C, a natural product biosynthetically related to Roc-A, displayed improved potency and therapeutic index against HCV infection. This study reveals a new class of HCV entry inhibitors that target the PHB1/2-CRaf pathway.

Topics: Antiviral Agents; Benzofurans; Cell Line; Drug Discovery; Hepacivirus; Hepatitis C; Humans; Prohibitins; Protein Binding; Proto-Oncogene Proteins c-raf; Repressor Proteins; Signal Transduction; Viral Envelope Proteins; Virus Internalization

To character a novel chimera(1b/2a) hepatitis C virus cell culture (HCVcc) system carrying envelope E1E2 coding gene from Hebei strain of China, chimera HCVcc (cHCVcc) was developed from Huh7.5-CD81 cells after transfection with in vitro transcribed full-length 1b/2a chimera RNA, which carrying envelope E1E2 coding gene from Hebei strain of China. Then the replication, expression and infectious titer of serial passage HCVcc were assessed by Real Time RT-PCR, indirect immunofluorescence assay (IFA) and Western blotting (WB). In addition, chimeric envelope gene from HCVcc was sequenced after serial passage. We found that the number of HCV positive focus increased gradually in cell post-transfection with chimera HCVcc (1b/2a) RNA and reach a peak platform (80% to 90%) at 41 days post-transfection; the expression of HCV protein was also confirmed by WAB during serial passage. At meantime, HCV RNA copy number in the supernatant peaked at 10(4)-10(7) copies/mL and the highest infectious titer of this 1b/2a cHCVcc reinfection were tested as 10(4) ffu/mL. Sequence analysis indicated 6 of adaptive amino acid substitutes occur among chimeric envelope E1E2 during serial passages. We con:luded that a novel 1b/2a chimera HCVcc carrying envelope E1E2 coding gene from Hebei strain of China was developed and its infectious titer increased after serial passage of HCVcc. This novel cHCVcc will be an effective tool for further evaluation of anti-virus drugs and immune effects against the major genotype from Chinese.

Topics: Cell Line; China; Hepacivirus; Hepatitis C; Humans; Serial Passage; Viral Envelope Proteins

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are important mediators for productive cell entry. However, knowledge about their structure, intra- or intermolecular dialogs, and conformational changes is scarce, limiting the design of therapeutic strategies targeting E1E2. Here we sought to investigate how certain domains of E1 and E2 have coevolved to optimize their interactions to promote efficient HCV entry. For this purpose we generated chimeric E1E2 heterodimers derived from two HCV 1a strains to identify and characterize crosstalk between their domains. We found an E1E2 combination that drastically impaired the infectivity of cell culture-derived HCV particles, whereas the reciprocal E1E2 combination led to increased infectivity. Using HCV pseudoparticle assays, we confirmed the opposing entry phenotypes of these heterodimers. By mutagenesis analysis, we identified a particular crosstalk between three amino acids of E1 and the domain III of E2. Its modulation leads to either a full restoration of the functionality of the suboptimal heterodimer or a destabilization of the functional heterodimer. Interestingly, we found that this crosstalk modulates E1E2 binding to HCV entry receptors SR-BI and CD81. In addition, we found for the first time that E1E2 complexes can interact with the first extracellular loop of Claudin-1, whereas soluble E2 did not. These results highlight the critical role of E1 in the modulation of HCV binding to receptors. Finally, we demonstrated that this crosstalk is involved in membrane fusion.. These results reveal a multifunctional and crucial interaction between E1 and E2 for HCV entry into cells. Our study highlights the role of E1 as a modulator of HCV binding to receptors and membrane fusion, underlining its potential as an antiviral target.

Topics: Amino Acid Sequence; Animals; Carcinoma, Hepatocellular; Claudin-1; Dimerization; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Liver Neoplasms; Membrane Fusion; Molecular Sequence Data; Protein Binding; Protein Structure, Tertiary; Rats; Scavenger Receptors, Class B; Tetraspanin 28; Viral Envelope Proteins

Hypervariable region 1 (HVR1) of envelope protein 2 (E2) of hepatitis C virus (HCV) serves important yet undefined roles in the viral life cycle. We previously showed that the viability of HVR1-deleted JFH1-based recombinants with Core-NS2 of H77 (H77(ΔHVR1), genotype 1a) and S52 (S52(ΔHVR1), genotype 3a) in Huh7.5 cells was rescued by E2 substitutions N476D/S733F and an E1 substitution, A369V, respectively; HVR1-deleted J6 (J6(ΔHVR1), genotype 2a) was fully viable. In single-cycle production assays, where HCV RNA was transfected into entry-deficient Huh7-derived S29 cells with low CD81 expression, we found no effect of HVR1 deletion on replication or particle release for H77 and S52. HCV pseudoparticle assays in Huh7.5 cells showed that HVR1 deletion decreased entry by 20- to 100-fold for H77, J6, and S52; N476D/S733F restored entry for H77(ΔHVR1), while A369V further impaired S52(ΔHVR1) entry. We investigated receptor usage by antibody blocking and receptor silencing in Huh7.5 cells, followed by inoculation of parental and HVR1-deleted HCV recombinants. Compared to parental viruses, scavenger receptor class B type I (SR-BI) dependency was decreased for H77(ΔHVR1/N476D/S733F), H77(N476D/S733F), S52(ΔHVR1/A369V), and S52(A369V), but not for J6(ΔHVR1). Low-density lipoprotein receptor (LDLr) dependency was decreased for HVR1-deleted viruses, but not for H77(N476D/S733F) and S52(A369V). Soluble LDLr neutralization revealed strong inhibition of parental HCV but limited effect against HVR1-deleted viruses. Apolipoprotein E (ApoE)-specific HCV neutralization was similar for H77, J6, and S52 viruses with and without HVR1. In conclusion, HVR1 and HVR1-related adaptive envelope mutations appeared to be involved in LDLr and SR-BI dependency, respectively. Also, LDLr served ApoE-independent but HVR1-dependent functions in HCV entry.

Topics: Cell Line, Tumor; Hepacivirus; Hepatitis C; Humans; Receptors, LDL; Receptors, Virus; Scavenger Receptors, Class B; Sequence Deletion; Viral Envelope Proteins; Virus Internalization

The identification of a specific immunogenic candidate that will effectively activate the appropriate pathway for neutralizing antibody production is fundamental for vaccine design. By using a monoclonal antibody (1H8) that neutralizes HCV in vitro, we have demonstrated here that 1H8 recognized an epitope mapped between residues A524 and W529 of the E2 protein. We also found that the epitope residues A524, P525, Y527 and W529 were crucial for antibody binding, while the residues T526, Y527 and W529 within the same epitope engaged in the interaction with the host entry factor CD81. Furthermore, we detected "1H8-like" antibodies, defined as those with amino acid-specificity similar to 1H8, in the plasma of patients with chronic HCV infection. The time course study of plasma samples from Patient H, a well-characterized case of post-transfusion hepatitis C, showed that "1H8-like" antibodies could be detected in a sample collected almost two years after the initial infection, thus confirming the immunogenicity of this epitope in vivo. The characterization of this neutralization epitope with a function in host entry factor CD81 interaction should enhance our understanding of antibody-mediated neutralization of HCV infections.

Topics: Amino Acid Motifs; Amino Acid Sequence; Antibodies, Monoclonal; Antibodies, Neutralizing; Binding Sites; Conserved Sequence; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Protein Binding; Tetraspanin 28; Viral Envelope Proteins

HCV is a worldwide health problem despite the recent advances in the development of more effective therapies. No preventive vaccine is available against this pathogen. However, non-sterilizing immunity has been demonstrated and supports the potential success of HCV vaccines. Induction of cross-neutralizing antibodies and T cell responses targeting several conserved epitopes, have been related to hepatitis C virus (HCV) clearance. Therefore, in this work, the immunogenicity of a preparation (MixprotHC) based on protein variants of HCV Core, E1, E2 and NS3 was evaluated in mice and monkeys. IgG from MixprotHC immunized mice and monkeys neutralized the infectivity of heterologous HCVcc. Moreover, strong CD4+ and CD8+ T cells proliferative and IFN-γ secretion responses were elicited against HCV proteins. Remarkably, immunization with MixprotHC induced control of viremia in a surrogate challenge model in mice. These results suggest that MixprotHC might constitute an effective immunogen against HCV in humans with potential for reducing the likelihood of immune escape and viral persistence.

Topics: Animals; Antibodies, Neutralizing; Chlorocebus aethiops; Female; Hepatitis C; Hepatitis C Antibodies; Immunity, Cellular; Immunoglobulin G; Interferon-gamma; Male; Mice; Mice, Inbred BALB C; T-Lymphocytes; Viral Core Proteins; Viral Envelope Proteins; Viral Hepatitis Vaccines; Viral Nonstructural Proteins

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and hepatocellular carcinoma. It infects human liver cells through several cellular protein receptors including CD81, SR-BI, claudin-1, and occludin. Previous reports also show that lectin receptors can mediate HCV recognition and entry. The envelope proteins of HCV (E1 and E2) are heavily glycosylated, further indicating the possible roles of lectin receptor-virus interaction in HCV infection. However, there is limited study investigating the relationship of HCV envelope glycoproteins and lectin as well as non-lectin receptors. Here we used surface plasmon resonance to examine the binding affinity of different glycoforms of recombinant HCV envelope protein to receptors and inspected the infectivity and assembly of HCV pseudoparticles composed of different glycoforms of envelope proteins. Our results indicated that DC-SIGN, L-SIGN, and Langerin had higher affinity to recombinant HCV envelope proteins in the presence of calcium ions than non-lectin receptors, and envelope proteins with Man8/9 N-glycans showed approximate 10-fold better binding to lectin receptors than envelope proteins with Man5 and complex type N-glycans. Interestingly, comparing among glycoforms, recombinant envelope proteins with Man5 N-glycans showed the highest binding affinity when interacting with non-lectin receptors. In summary, the glycans on HCV envelope protein play a modulatory role in HCV assembly and infection and direct HCV-receptor interaction, which mediates viral entry in different cells. Receptors with high affinity to HCV envelope proteins may be considered as targets for development of a therapeutic strategy against HCV.

Topics: Antigens, CD; Cell Adhesion Molecules; Cell Line; Glycosylation; Hepacivirus; Hepatitis C; Host-Pathogen Interactions; Humans; Lectins, C-Type; Liver; Mannose-Binding Lectins; Polysaccharides; Protein Binding; Receptors, Cell Surface; Recombinant Proteins; Viral Envelope Proteins

The Japanese fulminant hepatitis-1 (JFH1)-based hepatitis C virus (HCV) infection system has permitted analysis of the complete viral replication cycle in vitro. However, lack of robust infection systems for primary, patient-derived isolates limits systematic functional studies of viral intrahost variation and vaccine development. Therefore, we aimed at developing cell culture models for incorporation of primary patient-derived glycoproteins into infectious HCV particles for in-depth mechanistic studies of envelope gene function. To this end, we first constructed a packaging cell line expressing core, p7, and NS2 based on the highly infectious Jc1 genotype (GT) 2a chimeric genome. We show that this packaging cell line can be transfected with HCV replicons encoding cognate Jc1-derived glycoprotein genes for production of single-round infectious particles by way of trans-complementation. Testing replicons expressing representative envelope protein genes from all major HCV genotypes, we observed that virus production occurred in a genotype- and isolate-dependent fashion. Importantly, primary GT 2 patient-derived glycoproteins were efficiently incorporated into infectious particles. Moreover, replacement of J6 (GT 2a) core, p7, and NS2 with GT 1a-derived H77 proteins allowed production of infectious HCV particles with GT 1 patient-derived glycoproteins. Notably, adaptive mutations known to enhance virus production from GT 1a-2a chimeric genomes further increased virus release. Finally, virus particles with primary patient-derived E1-E2 proteins possessed biophysical properties comparable to Jc1 HCVcc particles, used CD81 for cell entry, were associated with ApoE and could be neutralized by immune sera.. This work describes cell culture systems for production of infectious HCV particles with primary envelope protein genes from GT 1 and GT 2-infected patients, thus opening up new opportunities to dissect envelope gene function in an individualized fashion.

Topics: Antibodies, Monoclonal; Apolipoproteins E; Genetic Complementation Test; Glycoproteins; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Neutralization Tests; Phylogeny; RNA, Viral; Tetraspanin 28; Viral Envelope Proteins; Viral Hepatitis Vaccines; Virion; Virus Replication

The recent history of the hepatitis C virus (HCV) subtypes 1a and 1b in the central region of Argentina is hypothesized by phylogeographic reconstruction using coalescent based Bayesian analyses. Direct partial E2 sequences from HCV 1a and 1b infected patients attending different health-care centers of the country were analyzed. The inferred date of the most recent common ancestor (tMRCA) for HCV-1a was: 1962 (between 1943 and 1977) and for HCV-1b was earlier: 1929 (between 1895 and 1953). Diverse ancestral populations were inferred from both subtypes in Córdoba and in Buenos Aires cities and after that, HCV spread within and between larger cities and to other smaller cities. The analyses suggested that HCV-1b was dispersed first and it is currently in a stationary phase whereas HCV-1a was dispersed latter and it is still in a growth phase. Finally, as it was observed in the developed countries, while the transmission of HCV-1b appears to have been somehow prevented, the HCV-1a may still represent a concern in the public health. Further work should be carried out to address their current transmission rate (and its main transmission route) in the Argentinean population.

Topics: Argentina; Genotype; Geography; Hepacivirus; Hepatitis C; History, 20th Century; History, 21st Century; Humans; Molecular Sequence Data; Phylogeny; Phylogeography; Retrospective Studies; Sequence Analysis, DNA; Viral Envelope Proteins

L-ficolin is a soluble pattern recognition molecule expressed by the liver that contributes to innate immune defense against microorganisms. It is well described that binding of L-ficolin to specific pathogen-associated molecular patterns activates the lectin complement pathway, resulting in opsonization and lysis of pathogens. In this study, we demonstrated that in addition to this indirect effect, L-ficolin has a direct neutralizing effect against hepatitis C virus (HCV) entry. Specific, dose-dependent binding of recombinant L-ficolin to HCV glycoproteins E1 and E2 was observed. This interaction was inhibited by soluble L-ficolin ligands. Interaction of L-ficolin with E1 and E2 potently inhibited entry of retroviral pseudoparticles bearing these glycoproteins. L-ficolin also inhibited entry of cell-cultured HCV in a calcium-dependent manner. Neutralizing concentrations of L-ficolin were found to be circulating in the serum of HCV-infected individuals. This is the first description of direct neutralization of HCV entry by a ficolin and highlights a novel role for L-ficolin as a virus entry inhibitor.

Topics: Complement Pathway, Mannose-Binding Lectin; Ficolins; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Lectins; Liver; Protein Binding; Receptors, Pattern Recognition; Recombinant Proteins; Viral Envelope Proteins; Virulence; Virus Internalization

Hepatitis C virus (HCV) is a universal health issue and a significant risk factor leading to hepatocellular carcinoma. HCV has infected approximately 170 million individuals worldwide. It is a member of Flaviviridae with positive sense RNA genome. In the absence of any effective vaccine against HCV, pegylated interferon with ribavirin is the standard of treatment against HCV infection. In this study, sequence and structural analysis of envelope 2 (E2) protein was performed which was isolated from patients of HCV genotype 3a in Pakistan. Then, epitopes were predicted which were specific for both B-cells and T-cells. Later, conservancy of epitopes was checked with the HCV 3a and 1a sequences from different countries. A total of 6 conserved epitopes were found from extra-membranous regions of E2 protein. Presence of conserved epitopes in E2 protein generates the possibility that these epitopes can be used to elicit the immune response against HCV.

Topics: Amino Acid Sequence; Epitopes; Hepatitis C; Humans; Molecular Sequence Data; Pakistan; Sequence Homology, Amino Acid; Viral Envelope Proteins; Viral Hepatitis Vaccines

In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies.. Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover, structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall, our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins.

Topics: Amino Acid Sequence; Conserved Sequence; Hepacivirus; Hepatitis C; Humans; Models, Molecular; Protein Binding; Receptors, Virus; Scavenger Receptors, Class B; Sequence Alignment; Tetraspanin 28; Viral Envelope Proteins; Virus Internalization

A challenge for hepatitis C virus (HCV) vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412-423 is conserved and antibodies to 412-423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs) against 412-423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc) were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412-423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape variants, which can co-exist with lower potency or levels of neutralizing activities.

Topics: Amino Acids; Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antibodies, Viral; Conserved Sequence; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Epitopes; Hepacivirus; Hepatitis C; Humans; Immune Evasion; Mice; Mutagenesis, Site-Directed; Mutation; Reverse Transcriptase Polymerase Chain Reaction; Viral Envelope Proteins; Viral Hepatitis Vaccines

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2's interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421-645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50's ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.

Topics: Amino Acid Sequence; Antiviral Agents; Binding Sites; Cell Line, Tumor; Cell Survival; Crystallography, X-Ray; Genotype; Hepacivirus; Hepatitis C; Humans; Kinetics; Ligands; Models, Molecular; Molecular Sequence Data; Protein Binding; Recombinant Proteins; Structural Homology, Protein; Surface Plasmon Resonance; Tetraspanin 28; Thermodynamics; Viral Envelope Proteins; Virus Internalization

Severe liver disease caused by chronic hepatitis C virus is the major indication for liver transplantation. Despite recent advances in antiviral therapy, drug toxicity and unwanted side effects render effective treatment in liver-transplanted patients a challenging task. Virus-specific therapeutic antibodies are generally safe and well-tolerated, but their potential in preventing and treating hepatitis C virus (HCV) infection has not yet been realized due to a variety of issues, not least high production costs and virus variability. Heavy-chain antibodies or nanobodies, produced by camelids, represent an exciting antiviral approach; they can target novel highly conserved epitopes that are inaccessible to normal antibodies, and they are also easy to manipulate and produce. We isolated four distinct nanobodies from a phage-display library generated from an alpaca immunized with HCV E2 glycoprotein. One of them, nanobody D03, recognized a novel epitope overlapping with the epitopes of several broadly neutralizing human monoclonal antibodies. Its crystal structure revealed a long complementarity determining region (CD3) folding over part of the framework that, in conventional antibodies, forms the interface between heavy and light chain. D03 neutralized a panel of retroviral particles pseudotyped with HCV glycoproteins from six genotypes and authentic cell culture-derived particles by interfering with the E2-CD81 interaction. In contrast to some of the most broadly neutralizing human anti-E2 monoclonal antibodies, D03 efficiently inhibited HCV cell-to-cell transmission.. This is the first description of a potent and broadly neutralizing HCV-specific nanobody representing a significant advance that will lead to future development of novel entry inhibitors for the treatment and prevention of HCV infection and help our understanding of HCV cell-to-cell transmission.

Topics: Amino Acid Sequence; Animals; Camelids, New World; Cell Communication; Cell Line, Tumor; Cells, Cultured; Epitope Mapping; Epitopes; Genotype; Hepacivirus; Hepatitis C; Humans; Liver; Molecular Sequence Data; Single-Domain Antibodies; Viral Envelope Proteins; Virus Internalization

Nosocomial transmission events still play an important role in hepatitis C virus (HCV) spreading. Among most reported medical procedures involved in nosocomial transmission, endoscopy procedures remain controversial and might be underestimated.. The aim of the study was to investigate a case of nosocomial person-to-person transmission of HCV in an endoscopy unit.. An acute HCV infection was detected in a person that had undergone a colonoscopy after an HCV-infected patient. Serum samples from both persons were subjected to a molecular epidemiology study. The HCV NS5B genetic region was amplified and directly sequenced and the E1-E2 region was amplified, cloned and sequenced (20 clones per specimen). All sequences were subjected to phylogenetic analyses. A conventional epidemiological investigation was performed to determine the most likely cause of HCV transmission.. NS5B sequence analysis revealed that both persons were infected with closely related HCV-1b strains. Furthermore, phylogenetic analysis of E1-E2 sequences evidenced a direct transmission between patients. The epidemiological investigation pointed out to anesthetic procedures as the most likely source of HCV transmission. The index case, not having spontaneously cleared the infection 10 months after infection, required antiviral treatment, which resulted in a sustained virological response.. The molecular epidemiology study performed provided evidence of a person-to-person transmission of HCV during a colonoscopy procedure, and the anesthetic procedure was the most likely source of HCV transmission. This study highlights the importance of strictly following standard precautions by healthcare workers in order to prevent nosocomial HCV transmission.

Topics: Cluster Analysis; Colonoscopy; Cross Infection; Hepacivirus; Hepatitis C; Humans; Molecular Epidemiology; Molecular Sequence Data; Phylogeny; RNA, Viral; Sequence Analysis, DNA; Sequence Homology; Viral Envelope Proteins; Viral Nonstructural Proteins

Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets.

Topics: Genome, Human; Genomics; Hepacivirus; Hepatitis C; Humans; Protein Binding; Proteins; Reproducibility of Results; Tetraspanin 30; Viral Envelope Proteins; Virus Internalization

Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL.

Topics: Antiviral Agents; Binding Sites; Drug Design; Hepacivirus; Hepatitis C; Hepatocytes; Host-Pathogen Interactions; Humans; Ligands; Models, Molecular; Protein Binding; Tetraspanin 28; Viral Envelope Proteins

Hepatitis C virus (HCV) infection is a significant public health problem with over 170,000,000 chronic carriers and infection rates increasing worldwide. Chronic HCV infection is one of the leading causes of hepatocellular carcinoma which was estimated to result in ∼10,000 deaths in the United States in the year 2011. Current treatment options for HCV infection are limited to PEG-ylated interferon alpha (IFN-α), the nucleoside ribavirin and the recently approved HCV protease inhibitors telaprevir and boceprevir. Although showing significantly improved efficacy over the previous therapies, treatment with protease inhibitors has been shown to result in the rapid emergence of drug-resistant virus. Here we report the activity of two proteins, originally isolated from natural product extracts, which demonstrate low or sub-nanomolar in vitro activity against both genotype I and genotype II HCV. These proteins inhibit viral infectivity, binding to the HCV envelope glycoproteins E1 and E2 and block viral entry into human hepatocytes. In addition, we demonstrate that the most potent of these agents, the protein griffithsin, is readily bioavailable after subcutaneous injection and shows significant in vivo efficacy in reducing HCV viral titers in a mouse model system with engrafted human hepatocytes. These results indicate that HCV viral entry inhibitors can be an effective component of anti-HCV therapy and that these proteins should be studied further for their therapeutic potential.

Topics: Animals; Antiviral Agents; Cell Line; Chlorophyta; Disease Models, Animal; Genotype; Hepacivirus; Hepatitis C; Humans; Mice; Models, Molecular; Plant Lectins; Protein Binding; Protein Conformation; Rhodophyta; Viral Envelope Proteins; Viral Load; Virus Internalization; Virus Replication

It has been reported that monoclonal antibodies (MAbs) to the E1E2 glycoproteins may have the potential to prevent hepatitis C virus (HCV) infection. The protective epitopes targeted by these MAbs have been mapped to the regions encompassing amino acids 313-327 and 432-443. In this study, we synthesized these two peptides and tested the reactivity of serum samples from 336 patients, 210 of which were from Chronic Hepatitis C (CHC) patients infected with diverse HCV genotypes. The remaining 126 samples were isolated from patients who had spontaneously cleared HCV infection. In the chronic HCV-infected group (CHC group), the prevalence of human serum antibodies reactive to epitopes 313-327 and 432-443 was 24.29% (51 of 210) and 4.76% (10 of 210), respectively. In the spontaneous clearance group (SC group), the prevalence was 0.79% (1 of 126) and 12.70% (16 of 126), respectively. The positive serum samples that contained antibodies reactive to epitope 313-327 neutralized HCV pseudoparticles (HCVpp) bearing the envelope glycoproteins of genotypes 1a or 1b and/or 4, but genotypes 2a, 3a, 5 and 6 were not neutralized. The neutralizing activity of these serum samples could not be inhibited by peptide 313-327. Six samples (SC17, SC38, SC86, SC92, CHC75 and CHC198) containing antibodies reactive to epitope 432-443 had cross-genotype neutralizing activities. The neutralizing activity of SC38, SC86, SC92 and CHC75 was partially inhibited by peptide 432-443. However, the neutralizing activity of sample SC17 for genotype 4HCVpp and sample CHC198 for genotype 1b HCVpp were not inhibited by the peptide. This study identifies the neutralizing ability of endogenous anti-HCV antibodies and warrants the exploration of antibodies reactive to epitope 432-443 as sources for future antibody therapies.

Topics: Amino Acid Sequence; Amino Acids; Antibodies, Neutralizing; Antibodies, Viral; Antibody Formation; Antibody Specificity; Cells, Cultured; Demography; Epitopes; Female; Genotype; Hepatitis C; Hepatitis C, Chronic; Humans; Male; Middle Aged; Molecular Sequence Data; Neutralization Tests; Peptides; Sequence Alignment; Viral Envelope Proteins

The poor response to the combined antiviral therapy of pegylated alfa-interferon and ribavarin for hepatitis C virus (HCV) infection may be linked to mutations in the viral envelope gene E1E2 (env), which can result in escape from the immune response and higher efficacy of viral entry. Mutations that result in failure of therapy most likely require compensatory mutations to achieve sufficient change in envelope structure and function. Compensatory mutations were investigated by determining positions in the E1E2 gene where amino acids (aa) covaried across groups of individuals. We assessed networks of covarying positions in E1E2 sequences that differentiated sustained virological response (SVR) from non-response (NR) in 43 genotype 1a (17 SVR), and 49 genotype 1b (25 SVR) chronically HCV-infected individuals. Binary integer programming over covariance networks was used to extract aa combinations that differed between response groups. Genotype 1a E1E2 sequences exhibited higher degrees of covariance and clustered into 3 main groups while 1b sequences exhibited no clustering. Between 5 and 9 aa pairs were required to separate SVR from NR in each genotype. aa in hypervariable region 1 were 6 times more likely than chance to occur in the optimal networks. The pair 531-626 (EI) appeared frequently in the optimal networks and was present in 6 of 9 NR in one of the 1a clusters. The most frequent pairs representing SVR were 431-481 (EE), 500-522 (QA) in 1a, and 407-434 (AQ) in 1b. Optimal networks based on covarying aa pairs in HCV envelope can indicate features that are associated with failure or success to antiviral therapy.

Topics: Antiviral Agents; Genotype; Hepacivirus; Hepatitis C; Humans; Interferon-alpha; Models, Biological; Models, Statistical; Multivariate Analysis; Phylogeny; Ribavirin; Treatment Outcome; Viral Envelope Proteins

The envelope glycoprotein E2 of hepatitis C virus (HCV) contains several hypervariable regions. Interestingly, 2 regions of intragenotypic hypervariability within E2 have been described as being specific to HCV subtype 3a. Based on their amino acid position in E2, they were named HVR495 and HVR575. Here, we further investigated these regions in order to better understand their role in HCV infection.. Sequences of HCV envelope glycoproteins from Pakistani patients infected with subtype 3a were cloned and compared with other subtype 3a sequences. The entry functions and the sensitivity to antibody neutralization of selected HCV glycoprotein sequences were tested in the HCV pseudotyped particles (HCVpp) system. In addition, the cell-cultured HCV system (HCVcc) was also used to confirm some of the data obtained with the HCVpp system.. We observed interesting new features within HVR495 and HVR575 for several subtype 3a isolates. Indeed, changes in glycosylation sites were observed with the appearance of a new glycosylation site within HVR495. Importantly, HCVpp and HCVcc that contained this new HVR495 glycosylation site were less sensitive to antibody neutralization.. We identified a new glycosylation site within the HVR495 region of HCV subtype 3a that has a protective effect against antibody neutralization.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antibodies, Neutralizing; Cell Line; Epitopes; Glycosylation; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Mutation; Pakistan; RNA, Viral; Sequence Alignment; Sequence Analysis, DNA; Tetraspanin 28; Viral Envelope Proteins

Hepatitis C virus (HCV) is highly variable and associated with chronic liver disease. Viral isolates are grouped into seven genotypes (GTs). Accumulating evidence indicates that viral determinants in the core to NS2 proteins modulate the efficiency of virus production. However, the role of the glycoproteins E1 and E2 in this process is currently poorly defined. Therefore, we constructed chimeric viral genomes to explore the role of E1 and E2 in HCV assembly. Comparison of the kinetics and efficiency of particle production by intragenotypic chimeras highlighted core and p7 as crucial determinants for efficient virion release. Glycoprotein sequences, however, had only a minimal impact on this process. In contrast, in the context of intergenotypic HCV chimeras, HCV assembly was profoundly influenced by glycoprotein genes. On the one hand, insertion of GT1a-derived (H77) E1-E2 sequences into a chimeric GT2a virus (Jc1) strongly suppressed virus production. On the other hand, replacement of H77 glycoproteins within the GT1a-GT2a chimeric genome H77/C3 by GT2a-derived (Jc1) E1-E2 increased infectious particle production. Thus, within intergenotypic chimeras, glycoprotein features strongly modulate virus production. Replacement of Jc1 glycoprotein genes by H77-derived E1-E2 did not grossly affect subcellular localization of core, E2, and NS2. However, it caused an accumulation of nonenveloped core protein and increased abundance of nonenveloped core protein structures with slow sedimentation. These findings reveal an important role for the HCV glycoproteins E1 and E2 in membrane envelopment, which likely depends on a genotype-specific interplay with additional viral factors.

Topics: Cell Line; Chimera; Genotype; Hepacivirus; Hepatitis C; Humans; Viral Envelope Proteins; Virus Assembly

Hepatitis C virus (HCV) remains a challenging public health problem worldwide. The identification of viral variants establishing de novo infections and definition of the phenotypic requirements for transmission would facilitate the design of preventive strategies. We explored the transmission of HCV variants in three cases of acute hepatitis following needlestick accidents. We used single-genome amplification of glycoprotein E1E2 gene sequences to map the genetic bottleneck upon transmission accurately. We found that infection was likely established by a single variant in two cases and six variants in the third case. Studies of donor samples showed that the transmitted variant E1E2 amino acid sequences were identical or closely related to those of variants from the donor virus populations. The transmitted variants harbored a common signature site at position 394, within hypervariable region 1 of E2, together with additional signature amino acids specific to each transmission pair. Surprisingly, these E1E2 variants conferred no greater capacity for entry than the E1E2 derived from nontransmitted variants in lentiviral pseudoparticle assays. Mutants escaping the antibodies of donor sera did not predominate among the transmitted variants either. The fitness parameters affecting the selective outgrowth of HCV variants after transmission in an immunocompetent host may thus be more complex than those suggested by mouse models. Human antibodies directed against HCV envelope effectively cross-neutralized the lentiviral particles bearing E1E2 derived from transmitted variants. These findings provide insight into the molecular mechanisms underlying HCV transmission and suggest that viral entry is a potential target for the prevention of HCV infection.

Topics: Amino Acid Sequence; Animals; Female; Hepacivirus; Hepatitis C; Humans; Male; Mice; Molecular Sequence Data; Phylogeny; Sequence Alignment; Viral Envelope Proteins

A major challenge for antiviral treatment of hepatitis C virus (HCV) infection is viral resistance, potentially resulting from the high variability of HCV envelope glycoproteins and subsequent selection of strains with enhanced infectivity and/or immune escape.. We used a bioinformatics and functional approach to investigate whether E1/E2 envelope glycoprotein structure and function were associated with treatment failure in 92 patients infected with HCV genotype 1.. Bioinformatics analysis identified 1 sustain virological response (R)-related residue in E1 (219T) and 2 non-SVR (NR)-related molecular signatures in E2 (431A and 642V) in HCV genotype 1a. Two of these positions also appeared in minimal networks separating NR patients from R patients. HCV pseudoparticles (HCVpp) expressing 431A and 642V resulted in a decrease in antibody-mediated neutralization by pretreatment sera. 431A/HCVpp entry into Huh7.5 cells increased with overexpression of CD81 and SR-BI. Moreover, an association of envelope glycoprotein signatures with treatment failure was confirmed in an independent cohort (Virahep-C).. Combined in silico and functional analyses demonstrate that envelope glycoprotein signatures associated with treatment failure result in an alteration of host cell entry factor use and escape from neutralizing antibodies, suggesting that virus-host interactions during viral entry contribute to treatment failure.

Topics: Adult; Antibodies, Neutralizing; Antibodies, Viral; Antiviral Agents; Computational Biology; Female; Genotype; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Immune Evasion; Male; Middle Aged; Mutagenesis, Site-Directed; Mutation; Neutralization Tests; Ribavirin; Structure-Activity Relationship; Treatment Failure; Viral Envelope Proteins; Virus Internalization

Chronic hepatitis C virus (HCV) infection can persist even in the presence of a broadly neutralizing antibody response. Various mechanisms that underpin viral persistence have been proposed, and one of the most recently proposed mechanisms is the presence of interfering antibodies that negate neutralizing responses. Specifically, it has been proposed that antibodies targeting broadly neutralizing epitopes located within a region of E2 encompassing residues 412 to 423 can be inhibited by nonneutralizing antibodies binding to a less conserved region encompassing residues 434 to 446. To investigate this phenomenon, we characterized the neutralizing and inhibitory effects of human-derived affinity-purified immunoglobulin fractions and murine monoclonal antibodies and show that antibodies to both regions neutralize HCV pseudoparticle (HCVpp) and cell culture-infectious virus (HCVcc) infection albeit with different breadths and potencies. Epitope mapping revealed the presence of overlapping but distinct epitopes in both regions, which may explain the observed differences in neutralizing phenotypes. Crucially, we failed to demonstrate any inhibition between these two groups of antibodies, suggesting that interference by nonneutralizing antibodies, at least for the region encompassing residues 434 to 446, does not provide a mechanism for HCV persistence in chronically infected individuals.

Topics: Amino Acid Motifs; Amino Acid Sequence; Antibodies, Neutralizing; Cell Line; Epitope Mapping; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Molecular Sequence Data; Sequence Alignment; Viral Envelope Proteins

Hepatitis C virus glycoprotein E2 contains 18 conserved cysteines predicted to form nine disulfide pairs. In this study, a comprehensive cysteine-alanine mutagenesis scan of all 18 cysteine residues was performed in E1E2-pseudotyped retroviruses (HCVpp) and recombinant E2 receptor-binding domain (E2 residues 384 to 661 [E2(661)]). All 18 cysteine residues were absolutely required for HCVpp entry competence. The phenotypes of individual cysteines and pairwise mutation of disulfides were largely the same for retrovirion-incorporated E2 and E2(661), suggesting their disulfide arrangements are similar. However, the contributions of each cysteine residue and the nine disulfides to E2 structure and function varied. Individual Cys-to-Ala mutations revealed discordant effects, where removal of one Cys within a pair had minimal effect on H53 recognition and CD81 binding (C486 and C569) while mutation of its partner abolished these functions (C494 and C564). Removal of disulfides at C581-C585 and C452-C459 significantly reduced the amount of E1 coprecipitated with E2, while all other disulfides were absolutely required for E1E2 heterodimerization. Remarkably, E2(661) tolerates the presence of four free cysteines, as simultaneous mutation of C452A, C486A, C569A, C581A, C585A, C597A, and C652A (M+C597A) retained wild-type CD81 binding. Thus, only one disulfide from each of the three predicted domains, C429-C552 (DI), C503-C508 (DII), and C607-C644 (DIII), is essential for the assembly of the E2(661) CD81-binding site. Furthermore, the yield of total monomeric E2 increased to 70% in M+C597A. These studies reveal the contribution of each cysteine residue and the nine disulfide pairs to E2 structure and function.

Topics: Amino Acid Sequence; Cell Line; Conserved Sequence; Cysteine; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Protein Binding; Protein Folding; Viral Envelope Proteins; Virus Internalization

The human immunodeficiency virus (HIV) and hepatitis C virus (HCV) share the same transmission routes which lead to high coinfection rates. Among HIV-infected individuals such rates reached 21% in Argentina, being HCV-1a the most predominant subtype. In this work, 25 HCV subtype 1a (HCV-1a) strains from Argentinean patients coinfected with HIV were studied based on E2 and NS5A sequences. Phylogenetic analyses indicated that 12 strains were highly related to each other, constituting a highly supported (posterior probability = 0.95) monophyletic group that we called "M." The remaining HCV strains (group dispersed or "D") were interspersed along the phylogenetic trees. When comparing both groups of HCV-1a, 10 amino acid differences were located in functional domains of E2 and NS5A proteins that appeared to affect eventually the peptides binding to MHC-I molecules thus favoring immune escape and contributing to the divergence of HCV genotypes. Bayesian coalescent analyses for HCV-1a cluster M isolates indicated that the time to the most recent common ancestor (tMRCA) overlaps with the age estimated recently for the HIV-BF epidemic in Argentina. Furthermore, the genomic characterization based on pol gene analysis from HIV viremic patients showed that most HIV isolates from patients coinfected with HCV-1a cluster M were BF recombinants with identical recombination patterns. In conclusion, these results suggest the presence of an HCV-1a monophyletic cluster with a potential HIV co-transmission by phylogenetic analyses.

Topics: Adult; Argentina; Cluster Analysis; Genotype; Hepacivirus; Hepatitis C; HIV Infections; Humans; Male; Middle Aged; Molecular Epidemiology; Molecular Sequence Data; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Nonstructural Proteins

The history behind the production of clotting factor concentrates produced differences in the prevalence of Hepatitis C Virus (HCV) and other blood-borne infections in haemophilic patients. Prevalence rates of HCV infection up to 100% were reported in patients treated with concentrates before 1985. Conversely, nowadays, viral inactivation and recombinant technologies have effectively prevented transfusion-transmitted viral pathogens. Recently, new HCV infections in three young brothers were observed. In the absence of any other risk of transmission, their HIV/HCV coinfected uncle, who was living in the same house, was subject to study. Plasma samples of the four relatives were investigated in order to test whether the infections have a common source. A phylogenetic approach using the most variable (E2) viral sequences was carried out using samples from the four family members. The HCV sequences from the study resulted highly related, being those obtained from the uncle the most ancestral ones. Because of the chronological order in which the infections occurred and the relatedness of the sequences, an infection from the uncle to his nephews is the most likely explanation. Special cares must be applied in the case of household contact among members of a family with inherited bleeding disorders.

Topics: Adolescent; Adult; Child; Family; Hemophilia A; Hepacivirus; Hepatitis C; HIV Infections; Humans; Male; Needles; Phylogeny; Viral Envelope Proteins

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5-1.0 log(10) reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Cell Line; Disease Models, Animal; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Liver Transplantation; Mutation; Neutralization Tests; Pan troglodytes; RNA, Viral; Tetraspanin 28; Viral Envelope Proteins; Viral Load

This paper investigated the envelope protein E1/E2 quasispecies genetic characterization of 4 HCV positive sera (Genotype 1b: 274, 366, 383; Genotype 2a: 283) in China. Nucleotide acid was extracted and glycoprotein E1/E2 (191-764aa) coding genes were obtained by RT-PCR, positive clones were randomly selected for sequencing. The phylogenetic relationships and the homology of nucleotide and amino acid were analyzed based on E1/E2 coding genes, and some vital functional regions of E1/E2 were characterized. A total of 43 sequences (274: 10; 283: 12; 366: 13; 383: 8) were obtained showing high genetic heterogeneity in HVR1 and HVR2 regions, while sequences of the neutralizing epitopes, transmembrane domain I, II and N-terminal ectodomain were comparatively conservative. Single base (C) insertion mutation at nt1279 ( E1 region, aa313), resulting in a mutated E1 coding protein (beginning at aa 313) and interruption at N terminus (aa 398) of HVR1 region of E2, was dominant quasispecies sequence(11/12) found in serum 283 . This is the first report on E1/E2 quasispecies in Chinese HCV patients and this novel pattern of insertion mutation provides important information for further study on HCV pathogenesis and immune evasion.

Topics: Amino Acid Sequence; Base Sequence; DNA Mutational Analysis; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Mutagenesis, Insertional; Viral Envelope Proteins

The E2 envelope glycoprotein of hepatitis C virus (HCV) binds to the host entry factor CD81 and is the principal target for neutralizing antibodies (NAbs). Most NAbs recognize hypervariable region 1 on E2, which undergoes frequent mutation, thereby allowing the virus to evade neutralization. Consequently, there is great interest in NAbs that target conserved epitopes. One such NAb is AP33, a mouse monoclonal antibody that recognizes a conserved, linear epitope on E2 and potently neutralizes a broad range of HCV genotypes. In this study, the X-ray structure of AP33 Fab in complex with an epitope peptide spanning residues 412 to 423 of HCV E2 was determined to 1.8 Å. In the complex, the peptide adopts a β-hairpin conformation and docks into a deep binding pocket on the antibody. The major determinants of antibody recognition are E2 residues L413, N415, G418, and W420. The structure is compared to the recently described HCV1 Fab in complex with the same epitope. Interestingly, the antigen-binding sites of HCV1 and AP33 are completely different, whereas the peptide conformation is very similar in the two structures. Mutagenesis of the peptide-binding residues on AP33 confirmed that these residues are also critical for AP33 recognition of whole E2, confirming that the peptide-bound structure truly represents AP33 interaction with the intact glycoprotein. The slightly conformation-sensitive character of the AP33-E2 interaction was explored by cross-competition analysis and alanine-scanning mutagenesis. The structural details of this neutralizing epitope provide a starting point for the design of an immunogen capable of eliciting AP33-like antibodies.

Topics: Animals; Antibodies, Neutralizing; Crystallography, X-Ray; Epitopes; Hepatitis C; Mice; Models, Molecular; Mutagenesis; Tetraspanin 28; Viral Envelope Proteins; Viral Hepatitis Vaccines; Viral Proteins

Hepatitis C virus (HCV) is the major cause of chronic liver disease worldwide. There is evidence that neutralizing anti-HCV antibodies may find potential applications in novel prophylactic and therapeutic strategies. This paper describes the very high neutralization activity and unique biological features of two broadly cross-reactive and cross-neutralizing anti-HCV human monoclonal IgG1 derived from human monoclonal recombinant Fab fragments.

Topics: Antibodies, Monoclonal; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoglobulin Fab Fragments; Immunoglobulin G; Kinetics; Neutralization Tests; Viral Envelope Proteins

Tracing risk factors for acquiring hepatitis C virus (HCV) in an HCV-infected patient, the only identified risk was working at the same butcher's counter of a supermarket as another HCV-infected patient, using a common ham cutting machine, with frequent bleeding hand injuries. A phylogenetic analysis showed a high percentage of nucleotide homology between the two patients' strains.

Topics: Adult; Female; Hand Injuries; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Occupational Diseases; Phylogeny; RNA, Viral; Sequence Analysis, DNA; Sequence Homology; Viral Envelope Proteins; Viral Nonstructural Proteins

Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein has been implicated in virus neutralization and persistence. We deleted HVR1 from JFH1-based HCV recombinants expressing Core/E1/E2/p7/NS2 of genotypes 1 to 6, previously found to grow efficiently in human hepatoma Huh7.5 cells. The 2a(ΔHVR1), 5a(ΔHVR1), and 6a(ΔHVR1) Core-NS2 recombinants retained viability in Huh7.5 cells, whereas 1a(ΔHVR1), 1b(ΔHVR1), 2b(ΔHVR1), 3a(ΔHVR1), and 4a(ΔHVR1) recombinants were severely attenuated. However, except for recombinant 4a(ΔHVR1), viruses eventually spread, and reverse genetics studies revealed adaptive envelope mutations that rescued the infectivity of 1a(ΔHVR1), 1b(ΔHVR1), 2b(ΔHVR1), and 3a(ΔHVR1) recombinants. Thus, HVR1 might have distinct functional roles for different HCV isolates. Ultracentrifugation studies showed that deletion of HVR1 did not alter HCV RNA density distribution, whereas infectious particle density changed from a range of 1.0 to 1.1 g/ml to a single peak at ∼1.1 g/ml, suggesting that HVR1 was critical for low-density HCV particle infectivity. Using chronic-phase HCV patient sera, we found three distinct neutralization profiles for the original viruses with these genotypes. In contrast, all HVR1-deleted viruses were highly sensitive with similar neutralization profiles. In vivo relevance for the role of HVR1 in protecting HCV from neutralization was demonstrated by ex vivo neutralization of 2a and 2a(ΔHVR1) produced in human liver chimeric mice. Due to the high density and neutralization susceptibility of HVR1-deleted viruses, we investigated whether a correlation existed between density and neutralization susceptibility for the original viruses with genotypes 1 to 6. Only the 2a virus displayed such a correlation. Our findings indicate that HVR1 of HCV shields important conserved neutralization epitopes with implications for viral persistence, immunotherapy, and vaccine development.

Topics: Amino Acid Motifs; Animals; Cell Line; Genetic Variation; Genotype; Hepacivirus; Hepatitis C; Humans; Mice; Neutralization Tests; Viral Envelope Proteins

Hepatitis C virus (HCV) core protein has long been considered an attractive candidate for inclusion in a protective vaccine. However, this protein may hamper the development of systemic immune responses because of its immune suppressive properties. We previously reported that immune responses to HCV core protein could be efficiently induced by attenuated Salmonella carrying the HCV core protein, but not the HCV core DNA vaccine. To optimize the combination of the core protein and envelope protein 2 (E2) into a vaccine formulation to induce cellular immune responses and neutralizing antibodies, we constructed a plasmid containing two expression cassettes. One expression cassette was included to regulate the expression of HCV core protein by an inducible in vivo-activated Salmonella promoter, the other was included to regulate the expression of HCV E2 protein by the cytomegalovirus enhancer/promoter. Oral immunization of BALB/c mice with the attenuated Salmonella strain SL7207 carrying this plasmid efficiently induced HCV core and E2-specific cellular immune responses and antibodies. IgG purified from immunized mice could neutralize the infectivity of HCV pseudoparticles (HCVpp) of both the autologous Con 1 isolate and the heterologous H77 isolate, and cell culture produced HCV (HCVcc) of Con1-JFH1 chimera. These results indicated that this vaccine strategy can effectively deliver core and E2 protein to the immune system and provide a promising approach for the development of prophylactic and therapeutic vaccines against HCV infection.

Topics: Administration, Oral; Animals; Antibodies, Neutralizing; Cytomegalovirus; Epitopes; Genetic Vectors; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunity, Cellular; Interferon-gamma; Interleukin-4; Mice; Mice, Inbred BALB C; Neutralization Tests; Plasmids; Promoter Regions, Genetic; Salmonella; T-Lymphocytes, Cytotoxic; Viral Core Proteins; Viral Envelope Proteins; Viral Hepatitis Vaccines

To explore the feasibility of induction of neutralization antibodies against hepatitis C virus (HCV) infection by HCV envelope 2 protein (E2) DNA vaccines immunization.. Two kinds of expression plasmids of HCV envelope 2 protein, plasmid pCI-1b661 Delta encoding hydrophobic carboxyl terminal truncated E2 and pCI-1b661 Delta encoding E2 with deletion of hypervariable region 1 (HVR1) and carboxyl terminal, were constructed and respectively transfeted 293T cells, and truncated E2 protein in whole cell lysate and supernatant of 293T cells were analyzed by Western blot. After BALB/c mouse were intramuscularly immunized by the plasmids, sera antibodies against HVR1 were detected by ELISA and the neutralization activity of the antibodies were assayed with HCV pseudotype particle (HCVpp).. Both plasmids could express secretary truncated E2 protein. All the mice immunized with plasmid pCI-1b661 produced HVR1 antibodies,while no HVR1 antibodies were detected in pCI-1b661 Delta immunized mice. The sera neutralization percentages against HCVpp in pCI1lb661 Delta and pCI-lb661 Delta immunized mice were (78.5 +/- 13.8)% and (38.7 +/- 6.5)%, respectively (P <0.01). Sera neutralization activity against HCVpp was positive correlated with the level of HVR1 antibodies in pCI-1b661 immunized mice (r = 0.967, P<0.01).. DNA vaccines expressing truncated E2 protein could induce neutralization antibodies against HCV, and neutralization antibodies mainly was consisted of the antibodies against HVR1.

Topics: Animals; Antibodies, Neutralizing; Hepacivirus; Hepatitis Antibodies; Hepatitis C; Immunization; Mice; Mice, Inbred BALB C; Vaccines, DNA; Viral Envelope Proteins; Viral Hepatitis Vaccines; Viral Proteins

Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antibody Specificity; Antigenic Variation; Carcinoma, Hepatocellular; Cell Line, Tumor; Conserved Sequence; Epitopes; Goats; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Neutralization Tests; Peptides; Vaccination; Vaccines, Subunit; Viral Envelope Proteins; Viral Hepatitis Vaccines; Viral Proteins

Hepatitis C virus (HCV) is a major health problem in developing countries including Pakistan. Chronic HCV infection results in progressive liver disease including fibrosis, cirrhosis, insulin resistance and eventually hepatocellular carcinoma (HCC). Ionotrophic purinergic (P2X) receptors are identified to involve in a spectrum of physiological and pathophysiological processes. However, the role of P2X receptors in HCV liver associated diseases still remains to be investigated. The current study was designed to identify the presence of P2X receptors in human liver cells. Furthermore, it investigates the response of P2X receptors towards HCV structural proteins (E1E2). To determine that how many isoforms of P2X receptors are expressed in human liver cells, human hepatoma cell line (Huh-7) was used. Transcripts (mRNA) of five different isoforms of P2X receptors were identified in Huh-7 cells. To examine the gene expression of identified isoforms of P2X receptors in presence of HCV structural proteins E1E2, Huh-7/E1E2 cell line (stably expressing HCV structural proteins E1E2) was used. The results showed significant increase (6.2 fold) in gene expression of P2X4 receptors in Huh-7/E1E2 cells as compared to control Huh-7 cells. The findings of present study confirmed the presence of transcripts of five different isoforms of P2X receptors in human liver cells and suggest that P2X4 receptors could be represented an important component of the purinergic signaling complex in HCV induced liver pathogenesis.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Gene Expression; Genotype; Hepacivirus; Hepatitis C; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Plasmids; Protein Isoforms; Real-Time Polymerase Chain Reaction; Receptors, Purinergic P2X4; RNA, Messenger; Signal Transduction; Transfection; Viral Envelope Proteins

HCV causes acute and chronic hepatitis which can eventually lead to permanent liver damage hepatocellular carcinoma and death. HCV glycoproteins play an important role in HCV entry by binding with CD81 receptors. Hence inhibition of virus at entry step is an important target to identify antiviral drugs against HCV.. The present study elaborated the role of CD81 and HCV glycoprotein E2 in HCV entry using retroviral pseudo-particles of 3a local genotype. Our results demonstrated that HCV specific antibody E2 and host antibody CD81 showed dose- dependent inhibition of HCV entry. HCV E2 antibody showed 50% reduction at a concentration of 1.5 ± 1 μg while CD81 exhibited 50% reduction at a concentration of 0.8 ± 1 μg. In addition, data obtained with HCVpp were also confirmed with the infection of whole virus of HCV genotype 3a in liver cells.. Our data suggest that HCV specific E2 and host CD81 antibodies reduce HCVpp entry and full length viral particle and combination of host and HCV specific antibodies showed synergistic effect in reducing the viral titer.

Topics: Antiviral Agents; Cell Line; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Liver; Tetraspanin 28; Viral Envelope Proteins; Virion; Virus Internalization

Topics: Adult; Cluster Analysis; Genotype; Hepacivirus; Hepatitis C; HIV Infections; Homosexuality, Male; Humans; Male; Middle Aged; Recurrence; RNA, Viral; Sequence Analysis, DNA; Sequence Homology; Viral Envelope Proteins; Viremia

In this study, we infected NOD/Scid/Jak3null mice engrafted human peripheral blood leukocytes (hu-PBL-NOJ) with measles virus Edmonston B strain (MV-Edm) expressing hepatitis C virus (HCV) envelope proteins (rMV-E1E2) to evaluate the immunogenicity as a vaccine candidate. Although human leukocytes could be isolated from the spleen of mock-infected mice during the 2-weeks experiment, the proportion of engrafted human leukocytes in mice infected with MV (10(3)-10(5)pfu) or rMV-E1E2 (10(4)pfu) was decreased. Viral infection of the splenocytes was confirmed by the development of cytopathic effects (CPEs) in co-cultures of splenocytes and B95a cells and verified using RT-PCR. Finally, human antibodies against MV were more frequently observed than E2-specific antibodies in serum from mice infected with a low dose of virus (MV, 10(0)-10(1)pfu, and rMV-E1E2, 10(1)-10(2)pfu). These results showed the possibility of hu-PBL-NOJ mice for the evaluation of the immunogenicity of viral proteins.

Topics: Animals; Blotting, Western; Callithrix; Cell Line; Coculture Techniques; Cytopathogenic Effect, Viral; Flow Cytometry; Hepatitis C; Humans; Leukocytes, Mononuclear; Measles virus; Mice; Mice, SCID; Polymerase Chain Reaction; Spleen; Vaccines, Synthetic; Viral Envelope Proteins; Viral Hepatitis Vaccines

The mechanism of the innate immune response to hepatitis C virus (HCV) has not been fully elucidated, largely due to the lack of an appropriate model. We used HCV transgenic (Tg) mice, which express core, E1, E2, and NS2 proteins regulated by the Cre/loxP switching expression system, to examine the innate immune response to HCV structural proteins. Twelve hours after HCV transgene expression, HCV core protein levels in Tg mouse livers were 15-47 pg/mg. In contrast, in Tg mice with a depletion of natural killer (NK) cells, we observed much higher levels of HCV core proteins (1,597 pg/ml). Cre-mediated genomic DNA recombination efficiency in the HCV-Tg mice was strongly observed in NK cell-depleted mice between 0.5 and 1 day as compared to non-treated mice. These data indicated that NK cells participate in the elimination of core-expressing hepatocytes in the innate immune responses during the acute phase of HCV infection.

Topics: Animals; Cytotoxicity, Immunologic; Hepacivirus; Hepatitis C; Hepatocytes; Immunity, Innate; Killer Cells, Natural; Liver; Mice; Mice, Inbred BALB C; Mice, Transgenic; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins

Development of an effective vaccine may be the key in the control of hepatitis C virus (HCV) infection. Recent studies have shown that HCV envelope proteins can induce broadly neutralizing antibodies against conserved domain for HCV binding to the cellular receptors. So HCV envelope proteins are considered as the major HCV vaccine candidate. In this study, we used Pichia pastoris yeast to express truncated HCV E1E2 protein, which consists of E1 residues 187-346 and E2 residues 381-699. The yeast can produce high level of recombinant HCV E1E2 protein. The protein has complex glycosylation and can bind to CD81, the putative HCV receptor. Moreover, the purified protein can efficiently induce anti-E1E2 antibodies in rabbits, which are able to neutralize two kinds of HCV pseudotype particles derived from HCV genotype 1a and 1b, as well as HCV virions derived from HCV genotype 2a. These findings indicate that the recombinant E1E2 glycoprotein is effective in inducing broadly neutralizing antibodies, and is a potent HCV vaccine candidate.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antigens, CD; Enzyme-Linked Immunosorbent Assay; Gene Expression; Glycosylation; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Neutralization Tests; Pichia; Plasmids; Rabbits; Recombinant Proteins; Tetraspanin 28; Viral Envelope Proteins; Viral Hepatitis Vaccines

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

Topics: Amino Acid Sequence; Antigens, CD; Antiviral Agents; Cells, Cultured; Drug Resistance, Viral; Drug Synergism; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Interferons; Molecular Sequence Data; Sequence Homology, Amino Acid; Small Molecule Libraries; Tetraspanin 28; Viral Envelope Proteins; Virus Internalization

To rational design HCV DNA vaccine candidates and evaluate their specific We design to construct two DNA vaccine candidates, one consists of immunity to HCV in mice.. We design to construct two DNA vaccine candidates, one consists of E2 (the envelope glycoprotein 2 of HCV) gene only, the second consists of E2-gAD (Globular Domain of Human Adiponectin) fusion gene via overlapping PCR. Confirm the expression of the DNA vaccines by Western blotting, and then vaccinated by injection of DNA vaccines with gene electrotransfer (GET) in BALB/c mice. The immune response was measured by IFN-gamma ELISPOT.. The DNA vaccine candidate consists of E2-gAD could effectively express in vitro , and it could induced a higher anti-HCV T cell response in mice than the one consists of E2 only.. The HCV DNA vaccine consists of E2-gAD fusion can increase the immunity of the E, to some extend, and the research paved a way to develop and optimize the novel HCV DNA vaccine.

Topics: Adiponectin; Animals; Female; Hepacivirus; Hepatitis C; Humans; Mice; Mice, Inbred BALB C; Recombinant Fusion Proteins; Vaccines, DNA; Viral Envelope Proteins; Viral Hepatitis Vaccines

Topics: Austria; Genotype; Hepacivirus; Hepatitis C; Host-Pathogen Interactions; Humans; Polymorphism, Genetic; RNA, Viral; Sequence Analysis, DNA; Viral Envelope Proteins

Acute hepatitis C virus (HCV) infection is rarely studied, but virus sequence evolution and host-virus dynamics during this early stage may influence the outcome of infection. Hypervariable region 1 (HVR1) is genetically diverse and under selective pressure from the host immune response. We analyzed HVR1 evolution by frequent sampling of an acutely infected HCV cohort.. Three or more pretreatment samples were obtained from each of 10 acutely infected subjects. Polymerase chain reaction amplification was performed with multiple primer combinations to identify the full range of sequences present. Positive samples were cloned and sequenced. Phylogenetic analyses were used to assess viral diversity.. Eight of the 10 subjects were coinfected with at least 2 HCV subtypes. Multiple subtypes were detected in individual samples, and their relative proportions changed through acute infection. The subjects with the most complex subtype structure also had a dynamic viral load; however, changes in viral load were not directly linked to changes in subtype.. This well-sampled cohort with acute HCV infection was characterized by dynamic coinfection with multiple viral subtypes, representing a highly complex virologic landscape extremely early in infection.

Topics: Adolescent; Adult; Austria; Cloning, Molecular; Female; Genotype; Hepacivirus; Hepatitis C; Host-Pathogen Interactions; Humans; Longitudinal Studies; Male; Middle Aged; Molecular Sequence Data; Polymorphism, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sequence Analysis, DNA; Viral Envelope Proteins

Hepatitis C is a major health problem affecting more than 200 million individuals in the world. Current treatment regimen consisting of interferon alpha and ribavirin does not always succeed in eliminating the virus completely from patient's body. One of the mechanisms by which virus evades the antiviral effect of interferon alpha involves protein kinase (PKR) eukaryotic initiation factor 2 alpha (eIF2a) phosphorylation homology domain (PePHD). This domain in genotype 1 strains is reportedly homologous to PKR and its target eIF2a. By binding to PKR, PePHD inhibits its activity and therefore cause virus to evade antiviral activity of interferon (IFN). Many studies have correlated substitutions in this domain to the treatment response and lead to inconclusive results. Some studies suggested that substitutions favor response while others emphasized that no correlation exists. In the present study we therefore compared sequences of PePHD domain of thirty one variants of six hepatitis C virus patients of genotype 3. Three of our HCV 3a infected patients showed rapid virological response to interferon alpha and ribavirin combination therapy whereas the remaining three had breakthrough to the same combination therapy. It is found that PePHD domain is not entirely conserved and has substitutions in some isolates irrespective of the treatment response. However substitution of glutamine (Q) with Leucine (L) in one of the breakthrough responders made it more identical to HCV genotype 1a. These substitutions in the breakthrough responders also tended to increase average hydrophilic activity thus making binding of PePHD to PKR and inhibition of PKR more favorable.

Topics: Amino Acid Sequence; Antiviral Agents; Drug Therapy, Combination; Eukaryotic Initiation Factor-2; Genotype; Hepacivirus; Hepatitis C; Humans; Interferons; Mutation; Pakistan; Protein Structure, Tertiary; Ribavirin; Sequence Alignment; Viral Envelope Proteins

Although hepatitis C virus (HCV) has been shown to readily escape from virus-specific T and B cell responses, its effects on natural killer (NK) cells are less clear. Based on two previous reports that recombinant, truncated HCV E2 protein inhibits NK cell functions via crosslinking of CD81, it is now widely believed that HCV impairs NK cells as a means to establish persistence. However, the relevance of these findings has not been verified with HCV E2 expressed as part of intact virions. Here we employed a new cell culture system generating infectious HCV particles with genotype 1a and 2a structural proteins, and analyzed direct and indirect effects of HCV on human NK cells. Antibody-mediated crosslinking of CD16 stimulated and antibody-mediated crosslinking of CD81 inhibited NK cell activation and interferon gamma (IFN-gamma) production. However, infectious HCV itself had no effect even at titers that far exceeded HCV RNA and protein concentrations in the blood of infected patients. Consistent with these results, anti-CD81 but not HCV inhibited NK cell cytotoxicity. These results were independent of the presence or absence of HCV-binding antibodies and independent of the presence or absence of other peripheral blood mononuclear cell populations.. HCV 1a or 2a envelope proteins do not modulate NK cell function when expressed as a part of infectious HCV particles. Without direct inhibition by HCV, NK cells may become activated by cytokines in acute HCV infection and contribute to infection outcome and disease pathogenesis.

Topics: Antigens, CD; Hepatitis C; Hepatitis C Antibodies; Humans; Interferon-gamma; Killer Cells, Natural; Leukocytes, Mononuclear; Peptide Fragments; Tetraspanin 28; Viral Envelope Proteins

Hepatitis C virus (HCV) encodes two transmembrane glycoproteins E1 and E2 which form a heterodimer. E1 is believed to mediate fusion while E2 has been shown to bind cellular receptors. It is clear that HCV uses a multi-receptor complex to gain entry into susceptible cells, however key elements of this complex remain elusive. In this study, the role of a highly conserved RGE/RGD motif of HCV E2 glycoprotein in viral entry was examined. The effect of each substitution mutation in this motif was tested by challenging susceptible cell lines with mutant HCV E1E2 pseudotyped viruses generated using a lentiviral system (HCVpp). In addition to assaying infectivity, producer cell expression and HCVpp incorporation of HCV E2 proteins, CD81 binding profiles, and conformation of mutants were examined.. Based on these characteristics, mutants either displayed wt characteristics (high infectivity [> or = 90% of wt HCVpp], CD81 binding, E1E2 expression, and incorporation into viral particles and proper conformation) or very low infectivity (< or = 20% of wt HCVpp). Only amino acid substitutions of the 3rd position (D or E) resulted in wt characteristics as long as the negative charge was maintained or a neutral alanine was introduced. A change in charge to a positive lysine, disrupted HCVpp infectivity at this position.. Although most amino acid substitutions within this conserved motif displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, and incorporation into HCVpp. Our results suggest that although RGE/D is a well-defined integrin binding motif, in this case the role of these three hyperconserved amino acids does not appear to be integrin binding. As the extent of conservation of this region extends well beyond these three amino acids, we speculate that this region may play an important role in the structure of HCV E2 or in mediating the interaction with other factor(s) during viral entry.

Topics: Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Antigens, CD; Cell Line; Conserved Sequence; Gene Expression; Hepacivirus; Hepatitis C; Humans; Integrins; Molecular Sequence Data; Protein Binding; Sequence Alignment; Tetraspanin 28; Viral Envelope Proteins; Virus Internalization

Hepatitis C virus (HCV) presents several regions involved potentially in evading antiviral treatment and host immune system. Two regions, known as PKR-BD and V3 domains, have been proposed to be involved in resistance to interferon. Additionally, hypervariable regions in the envelope E2 glycoprotein are also good candidates to participate in evasion from the immune system. In this study, we have used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples obtained just before initiation of therapy and after 6 or/and 12 months of treatment were available. A range of 25-100 clones per patient, genome region and time sample were obtained. The predominant amino acid sequences for each time sample and patient were determined. Next, the sequences of the PKR-BD and V3 domains and the hypervariable regions from different time samples were compared for each patient. The highest levels of variability were detected at the three hypervariable regions of the E2 protein and, to a lower extent, at the V3 domain of the NS5A protein. However, no clear patterns of adaptation to the host immune system or to antiviral treatment were detected. In summary, although high levels of variability are correlated to viral adaptive response, antiviral treatment does not seem to promote convergent adaptive changes. Consequently, other regions must be involved in evasion strategies likely based on a combination of multiple mechanisms, in which pools of changes along the HCV genome could confer viruses the ability to overcome strong selective pressures.

Topics: Amino Acid Sequence; Antiviral Agents; Drug Resistance, Viral; Drug Therapy, Combination; Hepacivirus; Hepatitis C; Humans; Interferon alpha-2; Interferon-alpha; Molecular Sequence Data; Mutation; Recombinant Proteins; Ribavirin; Treatment Failure; Viral Envelope Proteins; Viral Nonstructural Proteins

Broadly neutralizing antibodies are commonly present in the sera of patients with chronic hepatitis C virus (HCV) infection. To elucidate possible mechanisms of virus escape from these antibodies, retrovirus particles pseudotyped with HCV glycoproteins (HCVpp) isolated from sequential samples collected over a 26-year period from a chronically infected patient, H, were used to characterize the neutralization potential and binding affinity of a panel of anti-HCV E2 human monoclonal antibodies (HMAbs). Moreover, AP33, a neutralizing murine monoclonal antibody (MAb) to a linear epitope in E2, was also tested against selected variants. The HMAbs used were previously shown to broadly neutralize HCV and to recognize a cluster of highly immunogenic overlapping epitopes, designated domain B, containing residues that are also critical for binding of viral E2 glycoprotein to CD81, a receptor essential for virus entry. Escape variants were observed at different time points with some of the HMAbs. Other HMAbs neutralized all variants except for the isolate 02.E10, obtained in 2002, which was also resistant to MAb AP33. The 02.E10 HCVpp that have reduced binding affinities for all antibodies and for CD81 also showed reduced infectivity. Comparison of the 02.E10 nucleotide sequence with that of the strain H-derived consensus variant, H77c, revealed the former to have two mutations in E2, S501N and V506A, located outside the known CD81 binding sites. Substitution A506V in 02.E10 HCVpp restored binding to CD81, but its antibody neutralization sensitivity was only partially restored. Double substitutions comprising N501S and A506V synergistically restored 02.E10 HCVpp infectivity. Other mutations that are not part of the antibody binding epitope in the context of N501S and A506V were able to completely restore neutralization sensitivity. These findings showed that some nonlinear overlapping epitopes are more essential than others for viral fitness and consequently are more invariant during earlier years of chronic infection. Further, the ability of the 02.E10 consensus variant to escape neutralization by the tested antibodies could be a new mechanism of virus escape from immune containment. Mutations that are outside receptor binding sites resulted in structural changes leading to complete escape from domain B neutralizing antibodies, while simultaneously compromising viral fitness by reducing binding to CD81.

Topics: Amino Acid Substitution; Antibodies, Monoclonal; Antibody Affinity; Antigens, CD; Binding Sites; Cell Line; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Mutagenesis, Site-Directed; Mutation; Neutralization Tests; Sequence Analysis, Protein; Tetraspanin 28; Viral Envelope Proteins

We evaluated the expression of hepatitis C virus (HCV) antigen on liver grafts by immunohistochemical staining (IHS) using IG222 monoclonal antibody (mAb) against HCV-envelope 2 (E2).. The study material was 84 liver biopsy specimens obtained from 28 patients who underwent living donor liver transplantation (LDLT) for HCV infection. The biopsy samples were examined histopathologically, and by IHS using IG222 mAb against HCV-E2. Serum HCV-RNA level was measured in all patients. The IHS grades were compared among the three groups classified according to the time elapsed from LDLT (at 1-30, 31-179 and > or =180 days post-LDLT) and among four post-transplant conditions, including acute cellular rejection (ACR).. Immunoreactivity to IG222 was detected in 78.6% of the specimens obtained during the first month after LDLT, and there were no significant differences on the IHS grades between the three groups classified according to the time elapsed from LDLT. The IHS grades were significantly stronger in definite recurrent HCV (n = 12) and probable recurrent HCV (n = 7) than in definite ACR (n = 7) and other complications (n = 8). There were no significant differences in serum HCV-RNA levels among the four post-transplant conditions. There was no significant correlation between the IHS grades using IG222 mAb and serum HCV-RNA levels when data of 84 liver biopsy specimens were analyzed.. Constant HCV-E2 expression was observed in liver biopsy specimens obtained 1 month or longer. The strong HCV-E2 expression on liver grafts were associated with recurrent hepatitis C after LDLT, but the serum HCV-RNA levels were not.

Topics: Antibodies, Monoclonal; Biopsy; Female; Hepacivirus; Hepatitis C; Humans; Immunohistochemistry; Liver; Liver Cirrhosis; Liver Transplantation; Living Donors; Male; Middle Aged; Recurrence; RNA, Viral; Severity of Illness Index; Time Factors; Viral Envelope Proteins

Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus of about 9.6 kb. Like all enveloped viruses, the HCV membrane fuses with the host cell membrane during the entry process and thereby releases the genome into the cytoplasm, initiating the viral replication cycle. To investigate the features of HCV membrane fusion, we developed an in vitro fusion assay using cell culture-produced HCV and fluorescently labeled liposomes. With this model we could show that HCV-mediated fusion can be triggered in a receptor-independent but pH-dependent manner and that fusion of the HCV particles with liposomes is dependent on the viral dose and on the lipid composition of the target membranes. In addition CBH-5, an HCV E2-specific antibody, inhibited fusion in a dose-dependent manner. Interestingly, point mutations in E2, known to abrogate HCV glycoprotein-mediated fusion in a cell-based assay, altered or even abolished fusion in the liposome-based assay. When assaying the fusion properties of HCV particles with different buoyant density, we noted higher fusogenicity of particles with lower density. This could be attributable to inherently different properties of low density particles, to association of these particles with factors stimulating fusion, or to co-flotation of factors enhancing fusion activity in trans. Taken together, these data show the important role of lipids of both the viral and target membranes in HCV-mediated fusion, point to a crucial role played by the E2 glycoprotein in the process of HCV fusion, and reveal an important behavior of HCV of different densities with regard to fusion.

Topics: Carcinoma, Hepatocellular; Cholesterol; Electroporation; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Hydrogen-Ion Concentration; Immunoenzyme Techniques; Immunoprecipitation; Indoles; Liposomes; Liver Neoplasms; Luciferases; RNA, Viral; Sphingomyelins; Transcription, Genetic; Tumor Cells, Cultured; Viral Envelope Proteins; Virion; Virus Internalization

Hepatitis C virus (HCV) is a major health problem worldwide, infecting an estimated 170 million people. In this study, we have employed a large data set of sequences (14,654 sequences from between 25 and 100 clone sequences per analyzed region and per patient) from 67 patients infected with HCV genotype 1 (23 subtype 1a and 44 subtype 1b). For all patients, a sample prior to combined therapy with alpha interferon plus ribavirin was available, whereas for some patients additional samples after 6 or 12 months of treatment were also available. Twenty-seven patients responded to treatment (12 subtype 1a and 15 subtype 1b) and forty patients did not respond to treatment (11 subtype 1a vs. 29 subtype 1b). Two regions of the HCV genome were analyzed, one compressing the hypervariable regions (HVR1, HVR2 and HVR3) of the envelope 2 glycoprotein and another one including the interferon sensitive determining region (ISDR) and the V3 domain of the NS5A protein. Previously (Cuevas, J.M., Torres-Puente, M., Jiménez-Hernández, N., Bracho, M.A., García-Robles, I., Wrobel, B., Carnicer, F., del Olmo, J., Ortega, E., Moya, A., González-Candelas, F., 2008b. Genetic variability of hepatitis C virus before and after combined therapy of interferon plus ribavirin. Plos One 3 (8), e3058), several amino acid positions in both regions analyzed were detected to be under positive selection. Here, we have compared the amino acid composition of each positively selected position between responder and non-responder patients for both subtypes. If we exclude some non-conclusive cases, no clear differences were detected in any case. In conclusion, identifying specific positions as completely discriminatory of treatment response seems to be a difficult task. Our results, in concordance with previous studies, suggest that HCV evasion strategies are more likely based on a global increased variability, which would yield combinations of mutations with an increased resistance, than on the fixation of specific amino acids conferring resistance to antiviral treatment or immune response. In this sense, the particular systemic response from each patient could play an essential role in determining the outcome of the antiviral treatment.

Topics: Amino Acid Sequence; Chi-Square Distribution; Cohort Studies; Complementarity Determining Regions; Drug Resistance, Viral; Hepacivirus; Hepatitis C; Humans; Interferon-alpha; Ribavirin; Selection, Genetic; Sequence Analysis, Protein; Viral Envelope Proteins; Viral Nonstructural Proteins

From 2002 to 2007, 1,590 individuals were enrolled in an active surveillance program conducted in Metro Cebu, Philippines, where the anti-HCV-positive rate was significantly and constantly high among injecting drug users (83%, 793/960; 71-88%), especially among those living in downtown (89%, 683/770; 87-100%), despite the extremely low percentage of anti-HIV-positives (0.34%, 3/874). Sampling areas were then enlarged nationwide and the number of samples increased to 2,645 at the end of 2007. A total of 444 samples were positive for HCV RNA. Phylogenetic analysis based on NS5B and E1-E2 regions revealed that the most dominant HCV subtype was 1a, and followed by 2b, 2a, and 1b, and that the HCV strains had the largest variety in Metro Manila and its vicinity (P < 0.01). Interestingly, subtype 1b was detected solely in Metro Manila, and four HCV strains collected in this area showed higher homology to specific foreign strains retrieved from the Genbank/EMBL/DDBJ database with bootstrap values of 68-95% comparing with other strains analyzed in this nationwide study. These data suggest that HCV strains may be introduced occasionally into the Philippines possibly through Metro Manila as a main entry point. Considering the fact that an HIV epidemic started primarily via contaminated needle sharing in Asia, the constantly high rate of HCV infections and the newly introduced foreign HCV strains in the absence of HIV epidemic warrant further investigation on HCV entry and spread for early detection of an HIV epidemic in the Philippines.

Topics: Cluster Analysis; Female; Genotype; Hepacivirus; Hepatitis C; HIV Infections; Humans; Male; Molecular Epidemiology; Molecular Sequence Data; Philippines; Phylogeny; Polymorphism, Genetic; Prevalence; Sequence Analysis, DNA; Sequence Homology; Substance Abuse, Intravenous; Viral Envelope Proteins; Viral Nonstructural Proteins

Although viral variability studies have focused traditionally on consensus sequences, the relevance of molecular clone sequences for studying viral evolution at the intra-host level is being increasingly recognized. However, for this approach to be reliable, RT-PCR artifacts do not have to contribute excessively to the observed variability. Molecular clone sequences were obtained from an in vitro transcript to estimate the maximum error rate associated to RT-PCR for the Hepatitis C virus (HCV) E1-E2 region. On average, the frequency of RT-PCR errors was one order of magnitude lower than the level of intra-host genetic variability observed in samples from an HCV outbreak. However, RT-PCR errors were not distributed evenly along the E1-E2 region and were concentrated heavily in the hypervariable region 2 (HVR 2). Although it is concluded that RT-PCR molecular clone sequences are reliable, these results warn against extrapolation of RT-PCR error rates to different genome regions. The data suggest that the RNA sequence context or secondary structure can determine the fidelity of in vitro transcription or reverse transcription. Potentially, these factors might also modify the fidelity of the viral polymerase.

Topics: Artifacts; Disease Outbreaks; Hepacivirus; Hepatitis C; Humans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Viral Envelope Proteins

To evaluate the virological and clinical events occurring during a 3-year follow-up in three patients who, after symptomatic acute hepatitis C (AHC), experienced subsequent episodes of HC virus (V)-related acute liver cell necrosis.. The three patients were investigated for viral variability in the core, E1/E2, and NS5b regions during different phases of infection, and a computer-assisted analysis of the variation of known predicted epitopes in the consensus sequence was performed.. The first patient showed numerous genetic variations, which may be related to the maintenance of a chronic HCV infection state and to episodes of liver disease exacerbation. The second patient showed minimal viral variations associated with apparent resolution of the infection, but the same virus isolate, based on phylogenetic analysis, produced a second acute episode after the occult phase. The third patient, after the resolution of AHC, manifested a second episode of HCV infection by a different HCV sub-genotype.. Episodes of HCV-related acute liver cell necrosis after AHC may be associated to different virological patterns, such as the establishment of a chronic HCV infection, a reactivation of an occult virus, or a reinfection by a different HCV genotype.

Topics: Adult; Epitopes; Genotype; Hepacivirus; Hepatitis C; Humans; Liver; Male; Necrosis; Phylogeny; Polymorphism, Genetic; Sequence Analysis, DNA; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins

More than 120 million people worldwide are chronically infected with hepatitis C virus (HCV), making HCV infection the leading cause of liver transplantation in developed countries. Treatment is limited, and efficacy depends upon the infecting strain and the initial viral load. The HCV envelope glycoproteins (E1 and E2) are involved in receptor binding, virus-cell fusion, and entry into the host cell. HCV infection proceeds by endosomal acidification, suggesting that fusion of the viral envelope with cellular membranes is a pH-triggered event. E2 consists of an amino-terminal ectodomain, an amphipathic helix that forms a stem region, and a carboxy-terminal membrane-associating segment. We have devised a novel expression system for the production of a secreted form of E2 ectodomain (eE2) from mammalian cells and performed a comprehensive biochemical and biophysical characterization. eE2 is properly folded, as determined by binding to human CD81, blocking of infection of cell culture-derived HCV, and recognition by antibodies from patients chronically infected with different genotypes of HCV. The glycosylation pattern, number of disulfide bonds, oligomerization state, and secondary structure of eE2 have been characterized using mass spectrometry, size exclusion chromatography, circular dichroism, and analytical ultracentrifugation. These results advance the understanding of E2 and may assist in the design of an HCV vaccine and entry inhibitor.

Topics: Amino Acid Sequence; Cell Line; Glycosylation; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Protein Conformation; Protein Structure, Tertiary; Recombinant Proteins; Viral Envelope Proteins

Recombinant adeno-associated viruses (rAAV) vectors have been shown to mediate long-term transgene expression in mice and nonhuman primates. We have adapted viral vector system based on two rAAV vectors, namely rAAV1 and rAAV2. We have generated rAAV vectors expressing the envelope glycoprotein (E1 and E2) derived from Chinese HCV patient (genotype 1b) and used these to immunize BALB/c mice. We detected the total antibody titer by IFA and neutralizing antibody (nAb) using in vitro HCV neutralizing assays based on HCV pseudotyped particles. Furthermore, IFN-gamma ELISpot assay was used to assess the T cellular response against HCV at 12 weeks after rAAV1-E1E2 immunization. We also analyzed HCV envelope glycoprotein expression in muscle of rAAV1-E1E2 immunized mice. Our data showed: (i) rAAV1 directed long-term expression of HCV genes in mice; (ii) immunized intramuscularly with a single dose of rAAV elicited durable and effective immune responses in mice; and (iii) Moreover, rAAV1-E1E2 induced higher total antibody and nAb titers than rAAV2-E1E2 did. These data suggest that rAAV1 vectors could stimulate robust, durable, and effective immune responses against HCV.

Topics: Animals; Antibodies, Viral; Dependovirus; Female; Genetic Vectors; Hepacivirus; Hepatitis C; Humans; Mice; Mice, Inbred BALB C; Vaccines, DNA; Viral Envelope Proteins; Viral Vaccines

Hepatitis C virus (HCV) infection is a major worldwide health problem. Our previous results showed that HCV evolved to gain the enhanced infectivity and altered buoyant density distribution during persistent infections in vitro. Here we showed that a point mutation I414T in HCV E2 was mainly responsible for these phenotypic changes. While the I414T mutation had no significant effect on HCV RNA replication and viral entry, it enhanced the production of infectious viral particles and decreased the dependency of viral entry on the levels of HCV receptors. Furthermore, we showed that the I414T mutation reduced the association of viral particles with low-density lipoprotein or very low-density lipoproteins during the virus secretion process, and the infection of the delipidated virus was more sensitive to the blockade by an anti-E2 neutralizing antibody and recombinant CD81 proteins. Our results provided more insights into understanding the roles of lipoprotein associations in HCV life cycle.

Topics: Antibodies, Neutralizing; Antigens, CD; Cell Line; Hepacivirus; Hepatitis C; Humans; Lipoproteins, LDL; Point Mutation; RNA, Viral; Tetraspanin 28; Viral Envelope Proteins; Virion; Virus Internalization

Currently, the development of effective diagnostic reagents as well as treatments against Hepatitis C virus (HCV) remains a high priority. In this study, we have described the development of an alive cell surface--Systematic Evolution of Ligands by Exponential Enrichment (CS-SELEX) technique and screened the functional ssDNA aptamers that specifically bound to HCV envelope surface glycoprotein E2. Through 13 rounds of selection, the CS-SELEX generated high-affinity ssDNA aptamers, and the selected ssDNA aptamer ZE2 demonstrated the highest specificity and affinity to E2-positive cells. HCV particles could be specifically captured and diagnosed using the aptamer ZE2. A good correlation was observed in HCV patients between HCV E2 antigen-aptamer assay and assays for HCV RNA quantities or HCV antibody detection. Moreover, the selected aptamers, especially ZE2, could competitively inhibit E2 protein binding to CD81, an important HCV receptor, and significantly block HCV cell culture (HCVcc) infection of human hepatocytes (Huh7.5.1) in vitro. Our data demonstrate that the newly selected ssDNA aptamers, especially aptamer ZE2, hold great promise for developing new molecular probes, as an early diagnostic reagent for HCV surface antigen, or a therapeutic drug specifically for HCV.

Topics: Animals; Antigens, CD; Aptamers, Nucleotide; Base Sequence; Binding Sites; Cell Line; Cell Membrane; Fluorescein-5-isothiocyanate; Genotype; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Kinetics; Mice; Microscopy, Fluorescence; Molecular Probes; Molecular Sequence Data; Protein Binding; RNA, Viral; SELEX Aptamer Technique; Tetraspanin 28; Viral Envelope Proteins; Virion

Hepatitis C virus (HCV) infection has been closely related to mixed cryoglobulinemia (MC). During HCV infection, cryoglobulins derive from the restricted expression of few germline genes as VH1-69, a subfamily highly represented in anti-HCV humoral response. Little is known about the self-reacting IgM component of the cryoprecipitate. In the present study, the IgM/K repertoire of an HCV-infected cryoglobulinemic patient was dissected by phage-display on well-characterized anti-HCV/E2 VH1-69-derived monoclonal IgG1/Kappa Fab fragments cloned from the same patient. All selected IgM clones were shown to react with the anti-HCV/E2 antibodies belonging to VH1-69 subfamily. More than 60% of selected clones showed a bias in VH gene usage, restricted to two VH subfamilies frequently described in autoimmune manifestations (VH3-23; VH3-21). Moreover, all selected clones showed an high similarity (>98.5%) to germline genes evidencing their natural origin. A possible hypothesis is that clones belonging to some subfamilies are naturally prone to react against other VH gene subfamilies, as VH 1-69. An antigen-driven stimulation of these subfamilies, and their overexpression as in HCV infection, could lead to a breaking of humoral homeostatic balance exposing the patients to the risk of developing autoimmune disorders.

Topics: Autoantibodies; Cloning, Molecular; Cryoglobulinemia; Hepacivirus; Hepatitis C; Humans; Immunoglobulin Fab Fragments; Immunoglobulin M; Sequence Analysis, DNA; Viral Envelope Proteins

We tested the hypothesis that HIV-related immunosuppression alters the host-hepatitis C virus (HCV) interaction, resulting in fewer amino acid-changing substitutions in HCV viral variants. Higher HCV RNA levels in persons coinfected with HIV compared with HCV infection alone suggest increased viral replication. If this increase is dependent on decreased immune selective pressure, then a reduced rate of nucleotide changes resulting in amino acid replacements (nonsynonymous changes, dN) would be expected.. We investigated HCV envelope sequences over time in 79 persons with chronic HCV infection who were HIV negative (group 1) or HIV positive with (group 3) or without (group 2) severe immunodeficiency. We amplified a 1026-nt region of the HCV genome, which encodes a portion of the envelope glycoproteins E1 and E2, including hypervariable region-1 for direct sequence analysis.. The overall divergence between paired sequences, dS, dN, and dN/dS, all showed no significant differences among the 3 groups.. By measuring nucleotide substitutions in HCV sequences over time, we found no significant differences in the genetic divergence between HCV-monoinfected control subjects and HIV/HCV-coinfected subjects with various levels of immunodeficiency as measured by CD4+ T-cell counts.

Topics: Adult; Female; Hepacivirus; Hepatitis C; HIV Infections; Humans; Immune Tolerance; Longitudinal Studies; Male; Middle Aged; Mutation, Missense; Polymorphism, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Sequence Homology; Viral Envelope Proteins

We have conducted a large sequence study of the E1-E2 and NS5A regions of the HCV, subtypes 1a and b, both in patients previously treated with interferon, and untreated patients, who later responded, or not, to a combination therapy based on interferon plus ribavirin. We have examined the role played by the number of positively selected sites on disease progression and its relationship with several variables such as patients' age, sex and their risk of acquiring the disease. We have detected three groups of patients that respond or not to combination therapy: responders of intermediate age, older non-responders and young non-responders, they possess an increasing average number of positively selected sites in the E1-E2 region, respectively. We conclude that the host's genetic factors play an important role in whether the disease is contained or becomes chronic.

Topics: Adult; Aged; Amino Acid Sequence; Amino Acid Substitution; Antiviral Agents; Female; Hepacivirus; Hepatitis C; Humans; Interferons; Male; Middle Aged; Molecular Sequence Data; Ribavirin; RNA, Viral; Selection, Genetic; Sequence Analysis, DNA; Treatment Outcome; Viral Envelope Proteins; Viral Nonstructural Proteins

Hemodialysis patients are at increased risk of hepatitis C virus (HCV) infection. The aim of this study was to investigate a HCV outbreak in a hemodialysis unit using epidemiological and molecular methods. Between April 2003 and October 2003, anti-HCV seronconversion was detected in four patients attending the unit. These cases were added to 10 patients already anti-HCV positive upon admission in the unit. All 14 anti-HCV patients were tested for HCV RNA and HCV genotype. NS5B and HVR1/ E2 genomic regions were amplified and sequenced in all HCV RNA positive patients and phylogenetic analysis was performed. Furthermore, clinical-epidemiological records obtained from all patients were examined. All four patients newly infected harbored genotype 2c. Genotype 2c was also detected in 2 of 10 patients already anti-HCV positive upon admission. Phylogenetic analysis showed that all newly HCV infected patients harbored very closely related viral isolates that clustered together with the 2c isolate found in one of the two 2c chronic infected patients. All HCV-2c infected patients had no other risk factors except hemodialysis. Three of four newly HCV-2c infected patients and the one HCV-2c chronically infected involved in the outbreak received dialysis on the same day and same shift but used different machines. The remaining HCV-2c newly infected patient and one of the above cited three received dialysis on the same day during different shifts but used the same machine. The outbreak was probably due to breaks of infection control procedures although a related-machine transmission cannot be excluded in one of the cases.

Topics: Cluster Analysis; Cross Infection; Disease Outbreaks; Genotype; Hepacivirus; Hepatitis C; Humans; Italy; Molecular Epidemiology; Phylogeny; Renal Dialysis; RNA, Viral; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins

Hepatitis C virus (HCV) is a major health problem worldwide, infecting an estimated 170 million people. The high genetic variability of HCV contributes to the chronicity of hepatitis C. Here, we report results from a large-scale sequence analysis of 67 patients infected with HCV genotype 1, 23 with subtype 1a and 44 with subtype 1b. Two regions of the HCV genome were analysed in samples prior to combined therapy with alpha interferon plus ribavirin, one compressing the hypervariable regions (HVR1, HVR2 and HVR3) of the E2 glycoprotein and another one including the interferon-sensitive determining region (ISDR) and the V3 domain of the NS5A protein. Genetic diversity measures showed a clear tendency to higher genetic variability levels in nonresponder patients to antiviral treatment than in responder patients, although highly disperse values were present within each response group for both subtypes. A more detailed analysis of amino acid composition revealed the presence of several subtype-specific variants in a few positions, but no discriminating positions between responder and nonresponder patients were detected. Our results also revealed that most amino acid positions were highly conserved, especially for subtype 1a. We conclude that the outcome of the antiviral treatment might depend not only on the nature of one or a few independent positions, but more likely on the combination of several positions along the HCV genome. Moreover, the own host's ability to generate an appropriate systemic response, in combination with the action of antivirals, is also likely to be essential for treatment outcome.

Topics: Amino Acid Substitution; Antiviral Agents; Conserved Sequence; Hepacivirus; Hepatitis C; Humans; Interferon-alpha; Molecular Sequence Data; Mutation, Missense; Ribavirin; RNA, Viral; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Nonstructural Proteins

The hepatitis C virus (HCV) E2 protein has been shown to block apoptosis and has been suggested to facilitate persistent infection of the virus. Here, we report that the anti-apoptotic activity of E2 is mediated by activation of nuclear factor kappa B (NF-kappaB) that directs expression of survival gene products such as tumor necrosis factor (TNF-alpha) receptor-associated factor 2 (TRAF2), X-chromosome-linked inhibitor of apoptosis protein (XIAP), FLICE-like inhibitory protein (FLIP), and survivin. Increased levels of these proteins were observed in HCV-infected cells and a cell line producing HCV E2 protein. The activation of NF-kappaB was mediated by HCV-E2-induced expression of the molecular chaperone glucose-regulated protein 94 (GRP94). Overexpression of GRP94 alone resulted in expression of anti-apoptotic proteins and blocked apoptosis induced by tumor-necrosis-related apoptosis-inducing ligand (TRAIL). Interestingly, increased levels of GRP94 were observed in cells supporting HCV proliferation that originated from liver tissues from HCV patients. Moreover, small interfering RNA (siRNA) knock-down of GRP94 nullified the anti-apoptotic activity of HCV E2.. These data indicate that HCV E2 blocks apoptosis induced by HCV infection and the host immune system through overproduction of GRP94, and that HCV E2 plays an important role in persistent HCV infection.

Topics: Adult; Aged; Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Female; Gene Expression; Hepatitis C; Humans; Inhibitor of Apoptosis Proteins; Liver; Male; Membrane Glycoproteins; Microtubule-Associated Proteins; Middle Aged; Molecular Chaperones; Neoplasm Proteins; NF-kappa B; RNA, Small Interfering; Survivin; TNF Receptor-Associated Factor 2; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Viral Envelope Proteins; X-Linked Inhibitor of Apoptosis Protein

Immunoglobulin (Ig) GM and KM allotypes-genetic markers of gamma and kappa chains, respectively-are associated with the outcome of hepatitis C virus (HCV) infection, but the underlying mechanisms are not well understood. We hypothesized that GM and KM allotypes could contribute to the outcome of HCV infection by influencing the levels of IgG antibodies to the HCV glycoproteins E1E2. We serologically allotyped 100 African American individuals with persistent HCV infection for GM and KM markers and measured anti-E1E2 antibodies. Subjects with the GM 1,17 5,13 phenotype had significantly higher levels of anti-E1E2 antibodies than subjects who lacked this phenotype (p = 0.008). Likewise, subjects with the KM 1-carrying phenotypes had higher levels of anti-E1E2 antibodies than subjects who lacked these phenotypes (p = 0.041). Median titers were fourfold higher in persons expressing both GM 1,17 5,13 and KM 1-carrying phenotypes compared with those who lacked these phenotypes (p = 0.011). Interactive effects of these GM-KM phenotypes were previously found to be highly significantly associated with spontaneous HCV clearance. Results presented here show that Ig allotypes contribute to the interindividual differences in humoral immunity to the HCV epitopes, a finding that may provide a mechanistic explanation for their involvement in the outcome of HCV infection.

Topics: Adult; Alleles; Antibody Formation; Black or African American; Epitopes; Female; Haplotypes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Heterozygote; Homozygote; Humans; Immunoenzyme Techniques; Immunoglobulin G; Immunoglobulin Gm Allotypes; Immunoglobulin Km Allotypes; Male; Middle Aged; Phenotype; Serologic Tests; Viral Envelope Proteins

Human immunodeficiency virus (HIV) may influence the outcome and natural history of hepatitis C virus (HCV) infection through an impact on acute HCV-specific T cell responses.. Fifty-five HIV-positive males with acute HCV infection were identified; monoinfected individuals (n = 8) were used for peripheral blood mononuclear cell comparison. In 14 coinfected and 8 HCV-monoinfected patients, HCV-specific T cell responses against a range of HCV antigens were assessed using interferon (IFN)-gamma enzyme-linked immunospot (ELISpot) and proliferation assays. E1/E2 region genetic diversity and the selection pressure on the virus were measured in 8 coinfected patients by use of cloned sequences over time.. HCV persisted in 52 (95%) coinfected individuals. HCV/HIV coinfection significantly reduced IFN-gamma ELISpot responses versus those in HCV-monoinfected individuals, especially against nonstructural proteins (1/10 vs. 5/8; P = .008). In coinfected patients, increased HCV genetic diversity was observed between the first and subsequent time points, with no evidence for positive selection in the E1/E2 region sequenced.. HIV coinfection is associated with increased rates of HCV persistence and a lack of critical CD4 T cell responses, with no evidence of immune selection pressure during early HCV infection. Loss of key cellular immune responses against HCV during acute disease may contribute to the failure of early host control of HCV in HCV/HIV-coinfected patients.

Topics: Adult; Aged; Antigens, Viral; Cell Proliferation; Cells, Cultured; Hepacivirus; Hepatitis C; HIV Infections; Host-Pathogen Interactions; Humans; Interferon-gamma; Leukocytes, Mononuclear; Longitudinal Studies; Male; Middle Aged; Polymorphism, Genetic; Sequence Analysis, DNA; T-Lymphocyte Subsets; Viral Envelope Proteins

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. HCV infection is associated with high morbidity and has become a major problem in public health. Until now, there has been no effective prophylactic or therapeutic vaccine. BCG, a live vaccine typically used for tuberculosis prevention, has been increasingly utilized as a vector for the expression of recombinant proteins that will induce specific humoral and cellular immune responses. In this study, recombinant BCG (rBCG) was engineered to express a HCV multi-epitope antigen CtEm, and HLA-A2.1 transgenic mice were immunized with rBCG-CtEm. High levels of specific anti-HCV antibodies targeted to mimotopes of HVR1 were detected in the serum. HCV-specific lymphocyte proliferation assay, cytokine determination and cytotoxicity assay indicated that HCV epitope-specific cellular immune responses were elicited in vitro. The rBCG-CtEm immunization conferred protection against infection with the recombinant vaccinia virus (rVV-HCV-CNS) in vivo. These results suggest that rBCG expressing multi-epitope antigen may serve as an effective vaccine against HCV infection.

Topics: Amino Acid Sequence; Animals; Cell Division; Cytokines; Cytotoxicity Tests, Immunologic; Epitopes; Genetic Vectors; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; HLA-A2 Antigen; Immunization; Lymphocytes; Mice; Mice, Transgenic; Molecular Sequence Data; Mycobacterium bovis; Recombinant Proteins; Spleen; Vaccines, Synthetic; Vaccinia; Vaccinia virus; Viral Envelope Proteins; Viral Hepatitis Vaccines; Viral Nonstructural Proteins

Mutations in several subgenomic regions of hepatitis C virus (HCV) have been implicated in influencing the response to interferon (IFN) therapy. Sequences within HCV NS5A (PKR binding domain [PKRBD], IFN sensitivity-determining region [ISDR], and variable region 3 [V3]) were analyzed for the pretreatment serum samples of 60 HCV genotype 1-infected patients treated with pegylated IFN plus ribavirin (1b, n = 47; 1a, n = 13) but with different treatment outcomes, those with sustained virologic responses (SVR; n = 36) or nonresponders (NR; n = 24). Additionally, the sequence of the PKR/eIF-2alpha phosphorylation homology domain (E2-PePHD) region was determined for 23 patients (11 SVR and 12 NR). The presence of > 4 mutations in the PKRBD region was associated with SVR (P = 0.001) and early virologic responses (EVR; 12 weeks) (P = 0.037) but not rapid virologic responses (4 weeks). In the ISDR, the difference was almost statistically significant (68% of SVR patients with mutations versus 45% without mutations; P = 0.07). The V3 region had a very high genetic variability, but this was not related to SVR. Finally, the E2-PePHD (n = 23) region was well conserved. The presence of > 4 mutations in the PKRBD region (odds ratio [OR] = 9.9; P = 0.006) and an age of < or = 40 years (OR = 3.2; P = 0.056) were selected in a multivariate analysis as predictive factors of SVR. NS5A sequences from serum samples taken after 1 month of treatment and posttreatment were examined for 3 SVR and 15 NR patients to select treatment-resistant viral subpopulations, and it was found that in the V3 and flanking regions, the mutations increased significantly in posttreatment sera (P = 0.05). The genetic variability in the PKRBD (> 4 mutations) is a predictive factor of SVR and EVR in HCV genotype 1 patients treated with pegylated IFN and ribavirin.

Topics: Adult; Age Factors; Alanine Transaminase; Base Sequence; Cluster Analysis; Female; Genetic Variation; Genotype; Hepacivirus; Hepatitis C; Humans; Interferons; Male; Middle Aged; Molecular Sequence Data; Mutation; Odds Ratio; Phylogeny; Prospective Studies; Protein Structure, Tertiary; Ribavirin; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Nonstructural Proteins

Detection and localization of Hepatitis C Virus (HCV) in liver tissue is useful for diagnostic purposes as well as to elucidate the mechanisms by which the virus participates in hepatocarcinogenesis. However, so far, a sensitive method for HCV detection at the cellular level is lacking. We describe here the application of a novel antibody, D4.12.9, developed against serum-derived HCV RNA-positive particles, for the detection of E2 proteins by immunohistochemistry in fixed, archived specimens including liver biopsies of HCV-infected patients and surgical specimens of hepatocellular carcinoma. We demonstrate that D4.12.9 is a powerful tool for sensitive and specific detection of HCV, independently of viral genotype. This approach has applications to diagnosis as well as exploratory pathological studies.

Topics: Antibodies, Monoclonal; Antibody Specificity; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Humans; Immunohistochemistry; Liver; Liver Cirrhosis; Liver Neoplasms; Reproducibility of Results; Sensitivity and Specificity; Viral Envelope Proteins

Investigation of the mechanisms underlying hepatitis C virus (HCV) envelope glycoprotein gene evolution will greatly assist rational development of broadly neutralizing antibody-based vaccines or vaccine components. Previously, comprehensive cross-genotype evolutionary studies of E1E2 have not been possible due to the paucity of full-length envelope gene sequences representative of all major HCV genotypes (1-6) deposited in international sequence databases. To address this shortfall, a full-length E1E2 clone panel, corresponding to genotypes of HCV that were previously under-represented, was generated. This panel, coupled with divergent isolates available via international sequence databases, was subjected to high-resolution methods for determining codon-substitution patterns, enabling a fine-scale dissection of the selective pressures operating on HCV E1E2. Whilst no evidence for positive selection was observed in E1, the E2 protein contained a number of sites under strong positive selection. A high proportion of these sites were located within the first hypervariable region (HVR1), and statistical analysis revealed that cross-genotype adaptive mutations were restricted to a subset of homologous positions within this region. Importantly, downstream of HVR1, a differential genotype-specific distribution of adaptive mutations was observed, suggesting that subtly different evolutionary pressures shape present-day genotype diversity in E2 outside HVR1. Despite these observations, it is demonstrated that purifying selection due to functional constraint is the major evolutionary force acting on HCV E1E2. These findings are important in the context of neutralizing-antibody vaccine targeting, as well as in contributing to our understanding of E1E2 function.

Topics: Amino Acid Sequence; Genetic Variation; Genotype; Hepacivirus; Hepatitis C; Hepatitis C, Chronic; Humans; Molecular Sequence Data; Phylogeny; Selection, Genetic; Sequence Analysis, DNA; Viral Envelope Proteins

Hepatitis C virus (HCV) is a major human pathogen that causes serious liver disease, including cirrhosis and hepatocellular carcinoma. The primary target cells of HCV are hepatocytes, and entry is restricted by interactions of the envelope glycoproteins, E1 and E2, with cellular receptors. E1 and E2 form noncovalently linked heterodimers and are heavily glycosylated. Glycans contribute to protein folding and transport as well as protein function. In addition, glycans associated with viral envelopes mask important functional domains from the immune system and attenuate viral immunogenicity. Here, we explored the role of N- and O-linked glycans on E2, which is the receptor binding subunit of the HCV envelope. We identified a number of glycans that are critical for viral entry. Importantly, we showed that the removal of several glycans significantly increased the inhibition of entry by sera from HCV-positive individuals. Only some of the glycans that affected entry and neutralization were also important for CD81 binding. Our results show that HCV envelope-associated glycans play a crucial role in masking functionally important regions of E2 and suggest a new strategy for eliciting highly neutralizing antibodies against this virus.

Topics: Animals; Antigens, CD; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Humans; Polysaccharides; Tetraspanin 28; Viral Envelope Proteins; Virus Internalization

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Epitopes; Glycosylation; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Polysaccharides; Viral Envelope Proteins

Little is known about the potential effects of insertions and deletions (indels) on the evolutionary dynamics of hepatitis C virus (HCV). In fact, the consequences of indels on antiviral treatment response are a field of investigation completely unexplored. Here, an extensive sequencing project was undertaken by cloning and sequencing serum samples from 25 patients infected with HCV subtype 1a and 48 patients with subtype 1b. For 23 patients, samples obtained after treatment with alpha interferon plus ribavirin were also available. Two genome fragments containing the hypervariable regions in the envelope 2 glycoprotein and the PKR-BD domain in NS5A were sequenced, yielding almost 16 000 sequences. Our results show that insertions are quite rare, but they are often present in biologically relevant domains of the HCV genome. Moreover, their frequency distributions between different time samples reflect the quasispecies dynamics of HCV populations. Deletions seem to be subject to negative selection.

Topics: Amino Acid Sequence; Antiviral Agents; Drug Therapy, Combination; Gene Deletion; Genes, Viral; Hepacivirus; Hepatitis C; Humans; Interferon-alpha; Molecular Sequence Data; Mutagenesis, Insertional; Ribavirin; Sequence Alignment; Spain; Species Specificity; Viral Envelope Proteins; Viral Nonstructural Proteins

Hepatitis C virus (HCV) is a major pathogenic factor of liver diseases. During HCV infection, interaction of the envelope protein E2 of the virion, with target cells, is a crucial process for viral penetration into the cell and its propagation. We speculate that such interaction may trigger early signalling events required for HCV infection.. Human liver cell line L-02 was treated with HCV E2. The kinase phosphorylation levels of mitogen-activated protein kinase (MAPK) signalling pathways in the treated cells were analyzed by Western blotting. The proliferation of the E2-treated cells was evaluated by MTT assay.. HCV E2 was shown to be an efficient activator for MAPK pathways. Levels of phosphorylation of upstream kinases Raf-1 and MEK1/2 were seen to be elevated following E2 treatment and similarly, phosphorylation levels of downstream kinases MAPK/ERK and p38 MAPK also increased in response to E2 treatment, and specificity of kinase activation by E2 was confirmed. E2-induced MAPK/ERK activation was inhibited by the MEK1/2 inhibitor U0126 in a concentration-dependent manner. Blockage of relevant cellular receptors reduced activation of Raf-1, MEK1/2, MAPK/ERK and p38 MAPK by E2, indicating efflux of the E2 signal from extracellular to the intracellular spaces. Thus, kinase cascades of MAPK pathways were continuously affected by E2 presence. Moreover, enhancement of cell proliferation by E2 appeared to be associated with the dynamic phosphorylation of MAPK/ERK and p38 MAPK.. These results suggest that MAPK signalling pathways triggered by E2 may be a potential target for prevention of HCV infection.

Topics: Antigens, CD; Butadienes; Cell Line; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Hepatitis C; Humans; Liver; MAP Kinase Signaling System; Nitriles; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Receptors, LDL; Tetraspanin 28; Viral Envelope Proteins

Transmission of hepatitis C virus (HCV) from infected health care workers to patients rarely occurs. In 2003, a cluster of patients with HCV infection was identified at a medical center in Israel. All patients had a common history of various surgical procedures performed during the period 2001-2003. All patients had been anesthetized by an anesthesiologist who was an injection drug user and was infected with genotype 2a HCV. Screening was initiated by the hospital to identify newly infected patients with HCV infection and to determine the source of the iatrogenic HCV infection outbreak using comparative molecular analysis of the HCV E1 and HCV E2 hypervariable regions (HVR1 and HVR2).. A total of 1200 patients who were anesthetized by the anesthesiologist (the related group) and 873 hospital personnel and patients anesthetized by other anesthetists (the unrelated group) were examined. Serum samples were screened for anti-HCV antibodies, HCV RNA, and genotype. Sequence analysis of HVR1 and HVR2 was performed after reverse-transcriptase polymerase chain reaction.. HCV type 2a was found in 33 patients in the related group but in only 1 patient in the unrelated group. The differences between the sequences isolated from the related group serum samples and the sequences isolated from genotype 2a control group serum samples (obtained from 15 patients) were highly statistically significant. The genetic distances from the anesthesiologist sequence were 1.4%-4.4% in the HVR1 and 0%-3% in the HVR2 in the related group serum samples, whereas in the HCV genotype 2a control group serum samples, the genetic distances were 22%-45% and 10%-35%, respectively.. Molecular analysis revealed sequence similarity of HVR1 and HVR2 in the related group, suggesting that the anesthesiologist with chronic HCV infection may have transmitted HCV to 33 patients.

Topics: Disease Outbreaks; Genotype; Hepacivirus; Hepatitis C; Iatrogenic Disease; Infectious Disease Transmission, Professional-to-Patient; Phylogeny; Viral Envelope Proteins

Virus-like particles (VLPs) are highly immunogenic and proven to induce protective immunity. The small surface antigen (HBsAg-S) of hepatitis B virus (HBV) self-assembles into VLPs and its use as a vaccine results in protective antiviral immunity against HBV infections. Chimeric HBsAg-S proteins carrying foreign epitopes allow particle formation and have the ability to induce anti-foreign humoral and cellular immune responses.. The insertion of the hypervariable region 1 (HVR1) sequence derived from the envelope protein 2 (E2) of hepatitis C virus (HCV) into the major antigenic site of HBsAg-S ('a'-determinant) resulted in the formation of highly immunogenic VLPs that retained the antigenicity of the inserted HVR1 sequence. BALB/c mice were immunized with chimeric VLPs, which resulted in antisera with anti-HCV activity. The antisera were able to immunoprecipitate native HCV envelope complexes (E1E2) containing homologous or heterologous HVR1 sequences. HCV E1E2 pseudotyped HIV-1 particles (HCVpp) were used to measure entry into HuH-7 target cells in the presence or absence of antisera that were raised against chimeric VLPs. Anti-HVR1 VLP sera interfered with entry of entry-competent HCVpps containing either homologous or heterologous HVR1 sequences. Also, immunizations with chimeric VLPs induced antisurface antigen (HBsAg) antibodies, indicating that HBV-specific antigenicity and immunogenicity of the 'a'-determinant region is retained.. A multivalent vaccine against different pathogens based on the HBsAg delivery platform should be possible. We hypothesize that custom design of VLPs with an appropriate set of HCV-neutralizing epitopes will induce antibodies that would serve to decrease the viral load at the initial infecting inoculum.

Topics: Amino Acid Sequence; Animals; Cell Line; Hepacivirus; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunization; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Recombinant Fusion Proteins; Viral Envelope Proteins; Viral Hepatitis Vaccines; Virion

We compared the E2-HVR1 region in HCV-1b positive B-NHL cases from a multicenter study with sequences from studies related to lymphoproliferative disorders and B cell compartmentalisation. We found rare and unique mutations both in B-NHL isolates and in cases with lymphoproliferative disorders and lymphocyte infection. These rare mutations could have an important effect on HVR1 region and, as a consequence, on the binding of E2 on CD81, with a possible implication for both antigenic stimulation and HCV entry. In conclusion, the HCV predominants circulating in B-NHL cases seem to be associated with clonal selection of rare variants.

Topics: Antigens, CD; Genetic Variation; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Humans; Italy; Lymphoma, B-Cell; Molecular Sequence Data; Selection, Genetic; Species Specificity; Tetraspanin 28; Viral Envelope Proteins

One way to dissect the antibody response to an invading microorganism is to clone the antibody repertoire from immune donors and subsequently characterize the specific antibodies. Recently, methodological advances have allowed investigations of neutralizing antibodies against hepatitis C virus (HCV) in vitro. We have investigated three human mAbs, previously isolated from an individual infected with HCV of genotype 2b, that are known to cross-react in a binding assay to the envelope E2 protein of genotypes 1a and 1b. We now report that two of them have a neutralizing activity with a breadth not previously observed. Indeed, mAbs 1:7 and A8 recognized E2 from all of the six major genotypes, and they neutralized retroviral pseudoparticles [HCV pseudoparticles (HCVpp)] carrying genetically equally diverse HCV envelope glycoproteins. Importantly, these antibodies were also able to neutralize the cell culture infectious HCV clone JFH-1 in vitro, with IC(50) values of 60 ng/ml and 560 ng/ml, respectively. The conformational epitopes of these two broadly reactive antibodies were overlapping yet distinct and involved amino acid residues in the 523-535 region of E2, known to be important for the E2-CD81 interaction. The third antibody clone, representing a dominant population in the initial screen for these antibodies, was less broadly reactive and was unable to neutralize the genotype 2a infectious clone JFH-1. Our results confirm at the clonal level that broadly neutralizing human anti-HCV antibodies can be elicited and that the region amino acids 523-535 of the HCV envelope glycoprotein E2 carries neutralizing epitopes conserved across all genotypes.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Cell Line; Combinatorial Chemistry Techniques; Conserved Sequence; Epitope Mapping; Epitopes; Gene Library; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Molecular Sequence Data; Neutralization Tests; Protein Engineering; Sequence Homology, Amino Acid; Transfection; Viral Envelope Proteins

Neutralizing antibody responses elicited during infection generally confer protection from infection. Hepatitis C virus (HCV) encodes two glycoproteins E1 and E2 that are essential for virus entry and are the major target for neutralizing antibodies. To assess whether both glycoproteins are required for the generation of a neutralizing antibody response, rodents were immunized with a series of glycoproteins comprising full length and truncated versions. Guinea pigs immunized with HCV-1 genotype 1a E1E2p7, E1E2 or E2 generated high titer anti-glycoprotein antibody responses that neutralized the infectivity of HCVpp and HCVcc expressing gps of the same genotype as the immunizing antigen. Less potent neutralization of viruses bearing the genotype 2 strain J6 gps was observed. In contrast, immunized mice demonstrated reduced anti-gp antibody responses, consistent with their minimal neutralizing activity. Immunization with E2 alone was sufficient to induce a high titer response that neutralized HCV pseudoparticles (HCVpp) bearing diverse glycoproteins and cell culture grown HCV (HCVcc). The neutralization titer was reduced 3-fold by the presence of lipoproteins in human sera. Cross-competition of the guinea pig anti-E1E2 immune sera with a panel of epitope mapped anti-E2 monoclonal antibodies for binding E2 identified a series of epitopes within the N-terminal domain that may be immunogenic in the immunized rodents. These data demonstrate that recombinant E2 and E1E2 can induce polyclonal antibody responses with cross-reactive neutralizing activity, supporting the future development of prophylactic and therapeutic vaccines.

Topics: Animals; Cell Culture Techniques; Cross Reactions; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Neutralization Tests; Rodentia; Viral Envelope Proteins

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and there is a pressing need for the development of a preventative vaccine as well as new treatments. It was recently demonstrated that the mouse monoclonal antibody (mAb) AP33 potently neutralizes infectivity of HCV pseudoparticles (HCVpp) carrying E1E2 envelopes representative of all of the major genotypes of HCV. This study determined the prevalence of human serum antibodies reactive to the region of HCV E2 recognized by AP33. Antibodies recognizing this region were present in less than 2.5 % of sera obtained from individuals with chronic HCV infection. A similar prevalence was found in a smaller cohort of individuals who had experienced an acute infection, suggesting that AP33-like antibodies do not play a major role in natural clearance of HCV infection. Sera exhibited different patterns of reactivity to a panel of peptides representing circulating variants, highlighting the presence of distinct epitopes in this region. Only two sera contained antibodies that could recognize a specific AP33-reactive peptide mimotope. AP33-like antibodies made a measurable contribution to the ability of these sera to inhibit E2-CD81 interaction, but not to the overall neutralization of cell entry. Together, these data show that antibodies to the AP33 epitope are not commonly generated during natural infection and that generation of such antibodies via vaccination may require modified immunogens to focus the generation of specific antibodies. Importantly, individuals harbouring AP33-like antibodies are an important potential source of human mAbs for future therapeutic development.

Topics: Antibody Formation; Antigens, CD; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Protein Binding; Tetraspanin 28; Viral Envelope Proteins

Hepatitis C virus (HCV) envelope genes encoding glycoproteins E1 and E2 exhibits a high degree of variability that gives rise to differing phenotypic traits; including alterations in receptor-binding affinity and immune recognition and escape. This study aims to elucidate the relationship of the evolutionary patterns for HCV envelope glycoproteins to viral persistence.. HCV quasispecies were characterized in specimens collected every two to six months from a cohort of acutely HCV-infected subjects. We evaluated two individuals who spontaneously cleared viremia and three individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2; including hypervariable region 1 (HVR1). To detect representative variants for sequencing thirty-three cloned cDNAs representing each specimen were assessed by a method that combined analysis of a single-stranded conformational polymorphism (SSCP) method and heteroduplex analysis (HDA). For each patient, the rates of both synonymous and nonsynonymous substitutions for the E1, HVR1 and E2 regions outside HVR1 were evaluated. The amino acid sequences and predicted antigenic profiles were analyzed.. The genetic diversity within HVR1 was consistently higher than that in the E1 and E2 regions outside HVR1 in individuals with persistent viremia, but did not change markedly over time in those with clearance of viremia. For individuals with persistent viremia, the rate of nonsynonymous substitutions within the HVR1 region predominated and gradually increased, compared to that in the E1 and E2 regions outside HVR1. By contrast, the rates of both nonsynonymous and synonymous substitutions for the E1 and E2 regions, including HVR1, were consistently lower in individuals with clearance of viremia. HVR1 had a higher antigenic variable and lower positive charge in subjects with persistent viremia. All cysteine residues and N-linked glycosylation sites, some of which were known to play a major role in protein folding and others play a role in HCV entry, were 100% conserved among the sequenced cloned cDNAs from the two outcome groups.. HCV persistence may be associated with positive selection pressures on HVR1, rather than functional constraints in the envelope region.

Topics: Acute Disease; Adult; Amino Acid Sequence; Evolution, Molecular; Female; Hepacivirus; Hepatitis C; Heteroduplex Analysis; Humans; Male; Molecular Sequence Data; Polymorphism, Single-Stranded Conformational; Viral Envelope Proteins

Successful clearance of hepatitis C virus (HCV) infection has been associated with strong cellular immune responses against viral antigens. However, although the magnitude of these responses is clearly important for viral eradication, more studies are needed to unravel the fine specificity of the protective anti-HCV immunity in infected patients. This was the aim of the present study. Overlapping peptides spanning the sequence of HCV E2 and NS4a proteins were used to stimulate T cells from patients with chronic hepatitis C divided into three groups: naïve patients, patients who exhibited sustained response to interferon (IFN)-alpha therapy and patients who failed to respond to the treatment. Interleukin-2 production by stimulated cells was measured in each case. Patients with sustained response to therapy had stronger responses to E2 peptides than nonresponders, whereas naïve patients demonstrated intermediate reactivity. In the case of NS4a, responses against peptides where similar in all groups of patients. Analysis of the peptides recognized by T cells showed that responses were broad and heterogeneous, and some immunodominant epitopes, preferentially recognized by patients exhibiting sustained response to treatment, were found. These results confirm the role of cellular immune responses in viral clearance, and stress the importance of immunodominant regions within HCV antigens. These viral sequences may represent valuable immunogens for preparation of therapeutic or prophylactic vaccines.

Topics: Adult; Aged; Amino Acid Sequence; Female; Hepacivirus; Hepatitis C; HLA-DR Antigens; Humans; Immunodominant Epitopes; Interleukin-2; Male; Middle Aged; Molecular Sequence Data; Peptide Fragments; RNA, Viral; T-Lymphocytes; Viral Envelope Proteins; Viral Nonstructural Proteins

In order to study the incidence of hepatitis C virus (HCV) infection in Tunisian haemodialysis patients and detect its nosocomial transmission, 395 patients were enrolled in a prospective study (November 2001-2003). HCV serological and virological status was determined initially using, respectively a third generation ELISA and an RT-PCR qualitative assay. The genotype of the HCV isolates was determined by sequencing NS5B region. The issue of nosocomial transmission was addressed by sequencing the HVR-1 region of the E2 gene. About 20% of the patients had anti-HCV antibodies and HCV-RNA was detected in 73% of the anti-HCV positive patients. Two cases of de novo HCV infection were identified in two dialysis centers, during virological follow-up of patients susceptible to HCV infection. The incidence of de novo HCV infection was 0.5%. Determining the genotypes in the first center disclosed that all HCV-positive patients were infected with genotype 1b; sequencing of the HVR-1 region of the E2 gene provided strong evidence that the isolate from the newly infected patient and another infected dialysis patient were closely related, confirming nosocomial contamination. The investigation of the second center is pending. Besides, one patient with negative HCV serology had detectable HCV-RNA at the beginning of the study. This case had HCV genotype 1b, two other infected dialysis patients in the same unit had HCV genotypes 4k and 3a; thus precluding nosocomial transmission. Thanks to molecular and phylogenetic methods, one case of nosocomial HCV transmission in haemodialysis was confirmed. Epidemiological investigation suggested nosocomial transmission via the medical and/or nursing staff.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cross Infection; Female; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Incidence; Male; Middle Aged; Renal Dialysis; Species Specificity; Tunisia; Viral Envelope Proteins; Viral Nonstructural Proteins

To determine the prevalence of hepatitis C virus (HCV) infection in B-cell lymphoma in Japan. HCV infection and type II (monoclonal IgM) cryoglobulinaemia (CG) may be involved in the pathogenesis of low-grade B-cell lymphoma (ML) in southern Europe.. Forty-five (11.3%) of 400 B-cell ML cases were HCV antibody (Ab) positive, which was significantly (P < 0.01) higher than the blood donors (2.5%). Among them, 28 diffuse large B-cell lymphoma (DLBCL) cases were included. In the primary sites, 10 (47.6%) of 21 splenic DLBCL and seven (23.3%) of 30 gastric DLBCL were HCV Ab positive, which were significantly (P < 0.05) higher than the myeloma cases (4.9%). HCV infection was rarely (4.2%) detected in 24 lymphoplasmacytic and salivary gland low-grade B-cell ML cases. Type II CG was detected in one myeloma case (3.5%) of 29 HCV+ B-cell ML. By real-time polymerase chain reaction, HCV RNA was detected in fresh tumour tissues of all 11 B-cell ML cases examined. Lymphoma cells were positive for the envelope HCV non-structural (NS)3 and envelope (E2) proteins in six of eight examined B-cell ML cases.. The rare incidence of type II CG is characteristic of Japanese HCV+ ML patients and may influence the low incidence of low-grade B-cell ML. HCV infection may play a role in lymphomagenesis of splenic and gastric DLBCL.

Topics: Adolescent; Adult; Aged; Child; Comorbidity; Cryoglobulinemia; Epstein-Barr Virus Infections; Female; Genotype; Hepacivirus; Hepatitis B; Hepatitis C; HTLV-I Infections; Humans; Incidence; Japan; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Male; Middle Aged; Prevalence; RNA, Viral; Viral Envelope Proteins; Viral Nonstructural Proteins

Hepatitis C virus (HCV) plays a major role in the induction of type II mixed cryoglobulinaemia (MCII). The role of HCV proteins and virus-host interaction in the pathogenesis of MC remains to be defined. To address this issue, we have characterized, in detail, the monoclonal IgM and the viral component of circulating immune complexes in eight patients with HCV-associated MCII. The proportion of HCV-RNA compartmentalized in the cryoprecipitate (CP) varied greatly (10-80% of total HCV-RNA). The complementary determining region (CDR)3 sequences of monoclonal immunoglobulin M (IgM) VH and VK genes were highly homologous to rheumatoid factor and to antibodies against HCV-E2. Furthermore, the CDR3 sequences in some of our MCII patients were highly similar to those described in HCV-positive patients with non-Hodgkin's lymphoma (NHL). From these results, it appears that, as in the case of NHL, the IgM-rheumatoid factor (RF) production in MCII patients is antigen driven, namely by E2. However, the limited number of mutations in VH and VK genes with respect to the germline and their distribution showed that the B-cell response in these cases was prevented from undergoing affinity maturation. Furthermore, in patients with monoclonal IgM and definite compartmentalization of HCV in either CP or supernatant, a highly homogeneous E2-hypervariable region (HVR)1 sequence distribution was found (90-100% identical clones), a feature of the quasispecies frequently associated with an impaired humoral immune response to HCV. These findings suggest that in patients with HCV-associated MCII, maturation of monoclonal B lymphocytes may be blocked in a primitive stage preventing serious damaging effects because of the auto-reactivity of their secreted immunoglobulins.

Topics: Aged; Amino Acid Sequence; Antigen-Antibody Complex; Complementarity Determining Regions; Cryoglobulinemia; Female; Hepatitis C; Humans; Immunoglobulin M; Immunoglobulin Variable Region; Male; Middle Aged; Mutation; RNA, Viral; Sequence Alignment; Viral Envelope Proteins

The mouse monoclonal antibody (MAb) AP33, recognizing a 12 amino acid linear epitope in the hepatitis C virus (HCV) E2 glycoprotein, potently neutralizes retroviral pseudoparticles (HCVpp) carrying genetically diverse HCV envelope glycoproteins. Consequently, this antibody and its epitope are highly relevant to vaccine design and immunotherapeutic development. The rational design of immunogens capable of inducing antibodies that target the AP33 epitope will benefit from a better understanding of this region. We have used complementary approaches, which include random peptide phage display mapping and alanine scanning mutagenesis, to identify residues in the HCV E2 protein critical for MAb AP33 binding. Four residues crucial for MAb binding were identified, which are highly conserved in HCV E2 sequences. Three residues within E2 were shown to be critical for binding to the rat MAb 3/11, which previously was shown to recognize the same 12 amino acid E2 epitope as MAb AP33 antibody, although only two of these were shared with MAb AP33. MAb AP33 bound to a panel of functional E2 proteins representative of genotypes 1-6 with higher affinity than MAb 3/11. Similarly, MAb AP33 was consistently more efficient at neutralizing infectivity by diverse HCVpp than MAb 3/11. Importantly, MAb AP33 was also able to neutralize the cell culture infectious HCV clone JFH-1. In conclusion, these data identify important protective determinants and will greatly assist the development of vaccine candidates based on the AP33 epitope.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antibody Affinity; Cell Line; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Neutralization Tests; Peptide Library; Protein Conformation; Viral Envelope Proteins

Passive immunotherapy is potentially effective in preventing reinfection of liver grafts in hepatitis C virus (HCV)-associated liver transplant patients. A combination of monoclonal antibodies directed against different epitopes may be advantageous against a highly mutating virus such as HCV. Two human monoclonal antibodies (HumAbs) against the E2 envelope protein of HCV were developed and tested for the ability to neutralize the virus and prevent human liver infection. These antibodies, designated HCV-AB 68 and HCV-AB 65, recognize different conformational epitopes on E2. They were characterized in vitro biochemically and functionally. Both HumAbs are immunoglobulin G1 and have affinity constants to recombinant E2 constructs in the range of 10(-10) M. They are able to immunoprecipitate HCV particles from infected patients' sera from diverse genotypes and to stain HCV-infected human liver tissue. Both antibodies can fix complement and form immune complexes, but they do not activate complement-dependent or antibody-dependent cytotoxicity. Upon complement fixation, the monoclonal antibodies induce phagocytosis of the immune complexes by neutrophils, suggesting that the mechanism of viral clearance includes endocytosis. In vivo, in the HCV-Trimera model, both HumAbs were capable of inhibiting HCV infection of human liver fragments and of reducing the mean viral load in HCV-positive animals. The demonstrated neutralizing activities of HCV-AB 68 and HCV-AB 65 suggest that they have the potential to prevent reinfection in liver transplant patients and to serve as prophylactic treatment in postexposure events.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Drug Evaluation, Preclinical; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Liver Transplantation; Mice; Molecular Sequence Data; Neutralization Tests; Recurrence; Sequence Analysis, DNA; Viral Envelope Proteins

Mutations in several subgenomic regions have been implicated in influencing response to interferon therapy; however, a comprehensive picture of Indian patients was lacking. Based on the viral load and clinical factors, 10 out of 15 patients were found to be complete responders, whereas 5 patients were nonresponders. The pretreatment viral RNA load of the patients was found to be between 5.20 and 6.13 log10 IU/ml, which eventually fell to 2.77 log10 IU/ml after 24 weeks of treatment, whereas in the case of nonresponders, the average was 5.38 log10 IU/ml. In order to study the influence of the hepatitis C virus genotype on the response to interferon therapy, the 5' untranslated region sequences of the samples were analyzed, which showed that genotype 3 patients responded better than genotype 1 patients. Additionally, the mutations in the interferon sensitivity-determining region (ISDR) of the NS5A protein and the double-stranded RNA-activated protein kinase-eukaryotic initiation factor 2 alpha phosphorylation homology domain (PePHD) of the E2 envelope protein, before and after treatment, were compared with nonresponder prototype J. Although, no clear correlation was found in the case of the mutated ISDR, some significant changes in residues were observed in the PePHD region, which could be helpful in understanding the molecular basis of resistance to therapy. Interestingly, analysis of the quasispecies variations showed a change in genotype in one sample during treatment, which might have contributed to the resistance. The results suggest that the mutations in different regions of the viral genome might have a concerted effect on the response to interferon therapy.

Topics: 5' Untranslated Regions; Adult; Amino Acid Sequence; Drug Resistance, Viral; Female; Genes, Viral; Genotype; Hepacivirus; Hepatitis C; Humans; India; Interferons; Male; Middle Aged; Molecular Sequence Data; Mutation; Phylogeny; Sequence Homology, Amino Acid; Viral Envelope Proteins; Viral Nonstructural Proteins

The assumption of a molecular clock for dating events from sequence information is often frustrated by the presence of heterogeneity among evolutionary rates due, among other factors, to positively selected sites. In this work, our goal is to explore methods to estimate infection dates from sequence analysis. One such method, based on site stripping for clock detection, was proposed to unravel the clocklike molecular evolution in sequences showing high variability of evolutionary rates and in the presence of positive selection. Other alternatives imply accommodating heterogeneity in evolutionary rates at various levels, without eliminating any information from the data. Here we present the analysis of a data set of hepatitis C virus (HCV) sequences from 24 patients infected by a single individual with known dates of infection. We first used a simple criterion of relative substitution rate for site removal prior to a regression analysis. Time was regressed on maximum likelihood pairwise evolutionary distances between the sequences sampled from the source individual and infected patients. We show that it is indeed the fastest evolving sites that disturb the molecular clock and that these sites correspond to positively selected codons. The high computational efficiency of the regression analysis allowed us to compare the site-stripping scheme with random removal of sites. We demonstrate that removing the fast-evolving sites significantly increases the accuracy of estimation of infection times based on a single substitution rate. However, the time-of-infection estimations improved substantially when a more sophisticated and computationally demanding Bayesian method was used. This method was used with the same data set but keeping all the sequence positions in the analysis. Consequently, despite the distortion introduced by positive selection on evolutionary rates, it is possible to obtain quite accurate estimates of infection dates, a result of especial relevance for molecular epidemiology studies.

Topics: Bayes Theorem; Disease Outbreaks; Evolution, Molecular; Hepacivirus; Hepatitis C; Humans; Molecular Epidemiology; Phylogeny; RNA, Viral; Viral Envelope Proteins

To study the amino acid substitutions in the carboxy (C)-terminal part of E2 protein and in the interferon (IFN) sensitivity determining region (ISDR) and their correlation with response to IFN and viral load in 85 hepatitis C virus (HCV)-1b-infected patients treated with IFN.. The C-terminal part of E2 (codons 617-711) including PKR/eIF2alpha phosphorylation homology domain (PePHD) and ISDR was sequenced in 85 HCV-1b-infected patients treated by IFN monotherapy.. The amino acid substitutions in PePHD detected only in 4 of 85 patients were not correlated either with response to IFN or with viral load. The presence of substitutions in a N-terminal variable region (codons 617-641) in the C-terminal part of E2 was significantly correlated with both small viral load (33.9% vs 13.8%, P = 0.0394) and sustained response to IFN (25.0% vs 6.9%, P = 0.0429). Four or more substitutions in ISDR were significantly correlated with both small viral load (78.6% vs 16.2%, P < 0.0001) and sustained response to IFN (85.7% vs 2.9%, P < 0.0001). In multivariate analysis, ISDR in nonstructural (NS) 5A (OR = 0.39, P < 0.0001) and N-terminal variable region (OR = 0.51, P = 0.039) was selected as the independent predictors for small viral load, and ISDR (OR = 39.0, P < 0.0001) was selected as the only independent predictor for sustained response.. The N-terminal variable region in the C-terminal part of E2 correlates with both response to IFN monotherapy and viral load and is one of the factors independently associated with a small viral load.

Topics: Adult; Aged; Amino Acid Sequence; Amino Acid Substitution; Antiviral Agents; Codon; DNA, Viral; eIF-2 Kinase; Female; Hepacivirus; Hepatitis C; Humans; Interferons; Male; Microbial Sensitivity Tests; Middle Aged; Molecular Sequence Data; Multivariate Analysis; Mutation; Phosphorylation; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Treatment Outcome; Viral Envelope Proteins; Viral Load

A large share of hepatitis C virus amino acid sequence variation is concentrated within two hypervariable regions located at the N-terminus of the E2 envelope glycoprotein (HVR1 and HVR2). Interhost and intrahost comparison of 391 E2 sequences derived from 17 subjects infected with HCV using amino acid entropy revealed clustering of amino acid variability at a third site (residues 431-466), which was termed HVR3. Genetic distance analysis supported the division of HVR3 into three subdomains (HVR3a, HVR3b, and HVR3c). Study of synonymous and nonsynonymous nucleic acid substitutions confirmed that HVR3a was subjected to strong intrahost-selective pressure. Physicochemical and antigenicity predictions, conservation of key residues, and molecular modeling were concordant with one another and further validated the proposed organization of HVR3. Taken together, these results are suggestive of a role for HVR3 in cell surface receptor binding and viral entry akin to that proposed for HVR1 and HVR2.

Topics: Base Sequence; Chemical Phenomena; Chemistry, Physical; DNA, Viral; Female; Genes, Viral; Genetic Variation; Hepacivirus; Hepatitis C; Hepatitis C Antigens; HIV Infections; HIV-1; Humans; Models, Molecular; Pregnancy; Pregnancy Complications, Infectious; Protein Conformation; Selection, Genetic; Viral Envelope Proteins

L-SIGN is a C-type lectin that is expressed on liver sinusoidal endothelial cells. Capture of Hepatitis C virus (HCV) by this receptor results in trans-infection of hepatoma cells. L-SIGN alleles have been identified that encode between three and nine tandem repeats of a 23 residue stretch in the juxtamembrane oligomerization domain. Here, it was shown that these repeat-region isoforms are expressed at the surface of mammalian cells and variably bind HCV envelope glycoprotein E2 and HCV pseudoparticles. Differences in binding were reflected in trans-infection efficiency, which was highest for isoform 7 and lowest for isoform 3. These findings provide a molecular mechanism whereby L-SIGN polymorphism could influence the establishment and progression of HCV infection.

Topics: Alleles; Carcinoma, Hepatocellular; Cell Adhesion Molecules; HeLa Cells; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Lectins, C-Type; Minisatellite Repeats; Protein Isoforms; Receptors, Cell Surface; Receptors, Virus; Transfection; Viral Envelope Proteins

Hepatitis C virus (HCV) particles in serum associate with lipoproteins (LPs), and the low-density lipoprotein receptor (LDLr) has been implicated in virus attachment and entry into cells. To clarify the basis of interactions between virus and LPs, we determined whether HCV interacts with human LPs via its envelope glycoprotein E2. The binding of serum-derived virus-like particles, HCV E2, and HCV E2-LP complexes to CD81 and LDLr was studied. Incubation of HCV E2 protein with human and bovine LPs (very low density, low density, and high density) enhanced the binding of both HCV E2 and LPs to CD4+ lymphoblastoid (MOLT-4) cells, foreskin fibroblasts, and hepatocytes. The binding of HCV E2 to MOLT-4 cells was not enhanced when it was preincubated with lipid-free apoprotein B, which suggests that E2 interacts with the lipid moiety of human lipoproteins. The LP interaction was specific for HCV E2--incubation of HIV gp120 with LPs did not enhance gp120 binding to MOLT-4 cells. The enhanced HCV E2 binding required expression of both human CD81 and LDLr. These data suggest that HCV E2 associates with LDL and that the resulting complex enhances binding of both ligands to cells, which may contribute to the finding that HCV-infected individuals have significantly lower levels of LDL than control subjects.

Topics: Antigens, CD; Enzyme-Linked Immunosorbent Assay; Hepacivirus; Hepatitis C; Humans; In Vitro Techniques; Lipoproteins; Receptors, LDL; Tetraspanin 28; Viral Envelope Proteins

A structural genomics approach is proposed for the development of new diagnostic kits. It combines molecular modelling, peptide synthesis and immunological tests. The preliminary step is the development of a reliable three-dimensional structure of an immunodominant protein of the target pathogenic organism using the various bioinformatic strategies that are now available to structural biologists. Once the protein structure is obtained, the most surface-exposed fragments with minimal sequence variability among the different strains reported in the genomic data bank are reproduced synthetically as linear peptides. These peptides are then tested for immunoreactivity with the plasma of infected patients to determine whether the synthetic molecules have antigenic activity and can therefore be used to detect infecting agents. This structurally driven selection of mimotopes was successfully performed for the human hepatitis C virus, as five peptides that specifically interact with the plasma of HCV-infected patients were identified solely on the basis of the three-dimensional structure predicted for the E2 homodimer of the la viral subtype. A similar approach could easily be extended to a large variety of immunogenic proteins from other pathogenic organisms.

Topics: Enzyme-Linked Immunosorbent Assay; Genes, Viral; Hepacivirus; Hepatitis C; Humans; Immune Sera; Models, Molecular; Molecular Sequence Data; Peptides; Selection, Genetic; Sequence Alignment; Species Specificity; Viral Envelope Proteins; Viral Proteins

The factors leading to spontaneous clearance of hepatitis C virus (HCV) or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance is key to the development of more effective therapeutic and prophylactic strategies. Here, using a sensitive neutralization assay based on infectious HCV pseudoparticles (HCVpp), we have studied the kinetics of humoral responses in a cohort of acute-phase patients infected during a single nosocomial outbreak in a hemodialysis center. The 17 patients were monitored for the spontaneous outcome of HCV infection for 6 months before a treatment decision was made. Blood samples were taken frequently (15 +/- 4 per patient). Phylogenetic analysis of the predominant virus(es) revealed infection by only one of two genotype 1b strains. While all patients seroconverted, their sera induced two opposing effects in HCVpp infection assays: inhibition and facilitation. Furthermore, the ability of sera to facilitate or inhibit infection correlated with the presence of either infecting HCV strain and divided the patients into two groups. In group 1, the progressive emergence of a relatively strong neutralizing response correlated with a fluctuating decrease in high initial viremia, leading to control of viral replication. Patients in group 2 failed to reduce viremia within the acute phase, and no neutralizing responses were detected despite seroconversion. Strikingly, sera of group 2, as well as naive sera, facilitated infection by HCVpp displaying HCV glycoproteins from different genotypes and strains, including those retrieved from patients. These results provide new insights into the mechanisms of viral persistence and immune control of viremia.

Topics: Acute Disease; Adult; Aged; Alanine Transaminase; Amino Acid Sequence; Cohort Studies; Female; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Middle Aged; Molecular Sequence Data; Sequence Alignment; Time Factors; Viral Envelope Proteins; Viremia; Virus Replication

We analyzed a hepatitis C virus (HCV) transmission case in the hemodialysis unit of a private clinic by sequencing two genome regions of virus isolates from a number of patients attending this unit and some external controls. The analysis of 337 nucleotides (nt) in the NS5B region did not provide enough resolution to ascertain which patients were actually involved in the outbreak and the potential source. Nevertheless, this region allowed the exclusion of several patients as putative sources of the transmission case based on their genotypes and phylogenetic relationships. On the other hand, the analysis of several 472-nt-long clone sequences per sample in a more rapidly evolving region of the HCV genome, coding for the envelope proteins and encompassing hypervariable region 1, allowed us to establish the existence of at least two independent transmission events involving two different source patients and three recipients. The direction of the transmissions was further corroborated by different measures of genetic variability within and among samples.

Topics: Cross Infection; Disease Outbreaks; Female; Hemodialysis Units, Hospital; Hepacivirus; Hepatitis C; Humans; Male; Molecular Epidemiology; Molecular Sequence Data; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Nonstructural Proteins

Hepatitis C virus (HCV) circulates in the bloodstream in different forms, including complexes with immunoglobulins and/or lipoproteins. To address the significance of such associations, we produced or treated HCV pseudoparticles (HCVpp), a valid model of HCV cell entry and its inhibition, with naïve or patient-derived sera. We demonstrate that infection of hepatocarcinoma cells by HCVpp is increased more than 10-fold by human serum factors, of which high-density lipoprotein (HDL) is a major component. Infection enhancement requires scavenger receptor BI, a molecule known to mediate HDL uptake into cells as well as HCVpp entry, and involves conserved amino acid positions in hypervariable region 1 (HVR1) of the E2 glycoprotein. Additionally, we show that the interaction with human serum or HDL, but not with low-density lipoprotein, leads to the protection of HCVpp from neutralizing antibodies, including monoclonal antibodies and antibodies present in patient sera. Finally, the deletion or mutation of HVR1 in HCVpp abolishes infection enhancement and leads to increased sensitivity to neutralizing antibodies/sera compared to that of parental HCVpp. Altogether, these results assign to HVR1 new roles which are complementary in helping HCV to survive within its host. Besides immune escape by mutation, HRV1 can mediate the enhancement of cell entry and the protection of virions from neutralizing antibodies. By preserving a balance between these functions, HVR1 may be essential for the viral persistence of HCV.

Topics: Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Complementarity Determining Regions; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Lipoproteins, HDL; Liver Neoplasms; Mutagenesis, Site-Directed; Point Mutation; Recombinant Proteins; Viral Envelope Proteins

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and hepatocellular carcinoma worldwide. The purpose of this study was to determine how the HCV structural proteins affect the dynamic structural and functional properties of hepatocytes and measure the extra-hepatic manifestations induced by these viral proteins. A transgenic mouse model was established by expressing core, E1 and E2 proteins downstream of a CMV promoter. HCV RNA was detected using RT-PCR in transgenic mouse model tissues, such as liver, kidney, spleen and heart. Expression of the transgene was analysed by real-time PCR to quantify viral RNA in different tissues at different ages. Immunofluorescence analysis revealed the expression of core, E1 and E2 proteins predominantly in hepatocytes. Lower levels of protein expression were detected in spleen and kidneys. HCV RNA and viral protein expression increased in the liver with age. Histological analysis of liver cells demonstrated steatosis in transgenic mice older than 3 months, which was more progressed with age. Electron microscopy analysis revealed alterations in nuclei, mitochondria and endoplasmic reticulum. HCV structural proteins induce a severe hepatopathy in the transgenic mouse model. These mice became more prone to liver and lymphoid tumour development and hepatocellular carcinoma. In this model, the extra-hepatic effects of HCV, which included swelling of renal tubular cells, were mild. It is likely that the HCV structural proteins mediate some of the histological alterations in hepatocytes by interfering with lipid transport and liver metabolism.

Topics: Age Factors; Animals; Carcinoma, Hepatocellular; Cell Nucleus; Disease Models, Animal; Endoplasmic Reticulum; Fatty Liver; Female; Hematologic Neoplasms; Hepatitis C; Hepatocytes; Kidney; Liver; Liver Neoplasms; Male; Mice; Mice, Transgenic; Mitochondria; Viral Core Proteins; Viral Envelope Proteins

A correlation between the heterogeneity of hypervariable region 1 (HVR1) of E2 glycoprotein (gp) and Hepatitis C virus (HCV) antibody profile was investigated. Of 6 patients studied two were in acute phase, two in chronic phase and two showed signs of long-time HCV infection, i.e. liver cirrhosis. All the patients exhibited a vigorous antibody response to viral proteins C, NS3, NS4 and NS5. An antibody response to HVR1 of E2 was found in one patient in acute phase and in one or two patients in chronic phase. Such a response was not found in the two patients with liver cirrhosis. Single-stranded conformation polymorphism (SSCP) and sequence analyses of HVR1 of E2 showed the lowest HVR1 heterogeneity in patients in acute phase and the highest one in those in chronic phase, while the long-time carriers of the virus showed an intermediate heterogeneity. This may reflect a specific interplay between the virus and immune system. The HVR1 heterogeneity may rise in the course of infection as a means of evading the immune pressure. Then, when an organism is unable to clear the virus, because the responses to HVR1 epitopes are weakened or exhausted, a population of less heterogeneous HVR1 variants may be established.

Topics: Adult; Amino Acid Sequence; Amino Acid Substitution; Female; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Polymorphism, Single-Stranded Conformational; RNA, Viral; Sequence Analysis, DNA; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins

The question of whether viruses persist after apparent clearance of infection remains unanswered. Here, we describe a patient with hypogammaglobulinemia whose acute hepatitis C virus (HCV) infection appeared to resolve after receipt of interferon therapy, relapse immediately, and then clear spontaneously--only to relapse after receipt of corticosteroid therapy, and clear again, 8.5 years later. Sequencing indicated that the viruses detected during each relapse were virtually identical, with the hypervariable region 1 of E2 appearing to be monoclonal, which is typical of patients with hypogammaglobulinemia. Nonstructural 5A sequences exhibited quasispecies diversity initially but, after 8.5 years, had become monoclonal. The prolonged period of negativity for HCV RNA followed by relapse suggests that HCV may persist in apparent sustained viral responders.

Topics: Adrenal Cortex Hormones; Adult; Agammaglobulinemia; Alanine Transaminase; Amino Acid Sequence; Antiviral Agents; Female; Hepacivirus; Hepatitis C; Humans; Interferons; Mutation; Polymorphism, Genetic; Recurrence; RNA, Viral; Sequence Analysis, DNA; Sequence Homology; Viral Envelope Proteins; Viral Nonstructural Proteins

Hepatitis C virus infects humans world-wide. The virus genome varies greatly and it has several genotypes. HCV infection is highly prevalent in Central Africa and Cameroon. Initial studies on the genetic variability of HCV showed infection with HCV genotypes 1, 2, and 4. We have now sequenced the NS5b and E2 regions of 156 HCV isolates collected from patients presenting for diagnosis in Yaounde and used the data to describe the distribution of HCV genotypes and subtypes in patients with hepatitis in Cameroon. Genotype 1 was more frequent than Genotypes 4 and 2. Genotypes 1 and 4 were highly heterogeneous, containing many subtypes described previously (1b, 1c, 1e, 1h, 1l, 4f, 4t, 4p, 4k) and unsubtyped groups. There was a systematic phylogenetic concordance between NS5b and E2 sequence clustering. The Genotype 2 sequences did not vary. Neither subject age nor gender influenced HCV distribution. HCV Genotypes 1 and 4 are very heterogeneous in Cameroon, perhaps due to ancient infections. The homogeneity of HCV Genotype 2 indicates its more recent introduction from western Africa.

Topics: Adult; Aged; Cameroon; Female; Genetic Variation; Genotype; Hepacivirus; Hepatitis C; Humans; Male; Middle Aged; Molecular Sequence Data; Phylogeny; Pregnancy; Pregnancy Complications, Infectious; RNA, Viral; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Nonstructural Proteins

Injection drug users represent the largest cohort of patients with established hepatitis C virus (HCV) infection as well as the group that is at highest risk for new infections. Most published studies have focused on the clinical consequences of established HCV infection and have not examined the consequences of new infection. The aim of the current study was to measure the virological consequences of HCV in patients with ongoing injection drug use that might pose a risk for new and/or for superinfection with additional strains of HCV. We examined the following groups: (a) those with resolved HCV infection with ongoing injection drug use, (b) those with chronic infection who continued to inject and (c) those with chronic infection who no longer injected. Our study demonstrated a spectrum of responses. The majority of patients appeared to be 'protected' from new infection. None of six patients with resolved infection had detectable HCV RNA by quantitative or qualitative PCR when followed for 1 year. Similarly, despite ongoing injection drug use, no patient with persistent infection had a 'switch' in HCV genotype indicative of possible superinfection. Virological analysis of HCV quasispecies to detect possible infection with new variants of HCV in patients with apparently 'stable' infection, indicated divergence of virus over time, divergence that was unrelated to injection drug behaviour. Thus, patients with ongoing or prior HCV infection appear to develop immunity that protects against further infection with HCV despite repeated exposure.

Topics: Adult; Aged; Evolution, Molecular; Female; Genotype; Hepacivirus; Hepatitis C; Heteroduplex Analysis; Humans; Male; Middle Aged; Phylogeny; Polymerase Chain Reaction; RNA, Viral; Substance Abuse, Intravenous; Viral Envelope Proteins

We have previously shown that hepatocytes exposed to hepatitis C virus (HCV) and human immunodeficiency virus (HIV) envelope proteins undergo apoptosis. In this article, we further elucidate the signaling mechanisms that mediate this effect. We found that, in human hepatocellular carcinoma (HepG2) cells, HCV E2 protein and HIV glycoprotein (gp) 120 significantly up-regulated the Fas ligand (FasL) and enhanced the formation of the Fas death-inducing signaling complex downstream of Fas receptor activation. Moreover, after stimulation with HCV E2 and HIV gp120, enhanced expression of caspases 2 and 7 and increased caspase 3 activity were observed. In addition, we showed up-regulation of the proapoptotic molecule Bid and its association with caspase 8 after treatment with these envelope proteins. We also found that HCV E2 and HIV gp120 induced a partial translocation of Bid to the mitochondria, which resulted in the release of cytochrome C and the apoptosis-inducing factor. Thus, the results of this study suggest that FasL and Bid play an important role in HCV and HIV envelope protein-induced apoptosis.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Cell Line, Tumor; Death Domain Receptor Signaling Adaptor Proteins; Fas Ligand Protein; fas Receptor; Hepacivirus; Hepatitis C; Hepatocytes; HIV; HIV Envelope Protein gp120; HIV Infections; Humans; Liver; Mitochondria, Liver; Peptide Fragments; Time Factors; Up-Regulation; Viral Envelope Proteins

Immune correlates of protection against hepatitis C virus (HCV) infection are not well understood. Here we investigated 2 naive and 6 immunized chimpanzees before and after intravenous challenge, 12 weeks after the last immunization, with 100 50% chimpanzee infectious doses (CID(50)) of heterologous genotype 1b HCV. Vaccination with recombinant DNA and adenovirus vaccines expressing HCV core, E1E2, and NS3-5 genes induced long-term HCV-specific antibody and T-cell responses and reduced peak viral load about 100 times compared with controls (5.91 +/- 0.38 vs. 3.81 +/- 0.71 logs, respectively). There was a statistically significant inverse correlation between peak viral loads and envelope glycoprotein 2 (E2)-specific antibody responses at the time of challenge. Interestingly, one vaccinee that had sterilizing immunity against slightly heterologous virus generated the highest level of E2-specific total and neutralizing antibody responses as well as strong NS3/NS5-specific T-cell proliferative responses. The other four vaccinees with low levels of E2-specific antibody had about 44-fold reduced peak viral loads but eventually developed persistent infections. In conclusion, vaccine-induced E2-specific antibody plays an important role in prevention from nonhomologous virus infection and may provide new insight into the development of an effective HCV vaccine.

Topics: Animals; Hepatitis C; Hepatitis C Antibodies; Interferon-gamma; Pan troglodytes; T-Lymphocytes; Vaccination; Viral Envelope Proteins; Viral Hepatitis Vaccines; Viremia

Hepatitis C virus (HCV) is the major causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, and can be involved in very long chronic infections up to 30 years or more. Therefore, it has been speculated that HCV possesses mechanisms capable of modulating host defense systems such as innate and adaptive immunity. To investigate this virus-host interaction, we generated HCV replicons containing various HCV structural proteins and then analyzed the sensitivity of replicon-containing cells to the apoptosis-inducing agent, TRAIL. TRAIL-induced apoptosis was monitored by cleavage of procaspase-3 and procaspase-9 as well as that of their substrate poly(ADP-ribose) polymerase. TRAIL-induced apoptosis was inhibited in cells expressing HCV E2. Moreover, expression of HCV E2 enhanced the colony forming efficiency of replicon-containing cells by 25-fold. Blockage of apoptosis by E2 seems to be related to inhibition of TRAIL-induced cytochrome c release from the mitochondria. Based on these results, we propose that E2 augments persistent HCV infection by blocking host-induced apoptosis of infected cells.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochromes c; Hepatitis C; Humans; Membrane Glycoproteins; Mitochondrial Proteins; Replicon; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Viral Envelope Proteins; Viral Proteins

Current treatments for hepatitis C infection have limited efficacy, and there is no vaccine available. The goal of this study was to compare the immune response to several immunization combinations against hepatitis C virus (HCV). Six groups of mice were immunized at weeks 0, 4, and 8 with different combinations of a candidate HCV vaccine consisting of 100 microg recombinant HCV core/E1/E2 (rHCV) DNA plasmid and/or 25 microg rHCV polyprotein and 50 microL Montanide ISA- 51. Four weeks after the last injection, all groups of mice were sacrificed and blood samples and spleens were collected for measuring the levels of specific HCV antibodies (total IgG, IgG1, and IgG2a). Cell proliferation and intracellular interferon-gamma were also measured. Among the groups of immunized mice, only the mice immunized with rHCV DNA plasmid, rHCV polyprotein, and montanide (group D) and mice immunized with rHCV polyprotein and montanide (group F) demonstrated a significant increase in the total IgG titer after immunization. IgG1 was the predominant antibody detected in both groups D and F. No IgG2a was detected in any of the groups. Proliferation assays demonstrated that splenocytes from group D and group C (rHCV DNA primed/rHCV polyprotein boost) developed significant anti-HCV proliferative responses. The combination of an rHCV DNA plasmid, rHCV polyprotein, and montanide induced a high antibody titer with a predominance of IgG1 antibodies and recognized the major neutralization epitopes in HVR1. In contrast, group C did not show an increase in anti-HCV antibodies, but did show a proliferative response.

Topics: Adjuvants, Immunologic; Animals; Cell Proliferation; Epitopes; Hepatitis C; Hepatitis C Antibodies; Immunoglobulin G; Injections, Intramuscular; Interferon-gamma; Interleukin-5; Male; Mannitol; Mice; Mice, Inbred BALB C; Models, Animal; Neutralization Tests; Oleic Acids; T-Lymphocytes; Vaccines, DNA; Viral Core Proteins; Viral Envelope Proteins; Viral Hepatitis Vaccines

The effect of highly active antiretroviral therapy (HAART) on HCV replication is controversial, with some studies reporting no effect and others increases, reductions and even clearances of HCV RNA after treatment. In this study, the effect of HAART was investigated on the titre of anti-HCV specific antibodies and on the relationship between these antibodies and HCV RNA level in a cohort of 24 patients with inherited bleeding disorders. A significant inverse correlation between antibodies to both total HCV proteins and HCV RNA (R = -0.42, P = 0.05) and between antibodies to HCV envelope glycoproteins and HCV RNA (R = -0.54, P = 0.01) was observed pre-HAART. The relationship disappeared or was obscured after therapy (R = 0.24, P = 0.30 and R = 0.16, P = 0.50, respectively). Thus, we show that HAART affects the HCV specific humoral immune responses without affecting the HCV RNA level.

Topics: Animals; Antiretroviral Therapy, Highly Active; Cells, Cultured; Hemophilia A; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C, Chronic; HIV Infections; HIV-1; Humans; Male; RNA, Viral; Spodoptera; Viral Envelope Proteins; Viral Load; Viral Structural Proteins

It is unknown whether all hepatitis C virus (HCV) quasispecies variants found within patient serum have equal capacity to associate with the liver after transplantation; however, in vitro models of HCV infection suggest that variations in the hypervariable region 1 (HVR1) of the second envelope protein (E2) may be important in infectivity. The hypothesis of the current study is that the two hypervariable regions (HVR1 and HVR2) within E2 are important in the initial virus-liver interaction, and, therefore, certain HCV quasispecies variants will be isolated from the liver after reperfusion. In 8 patients with end-stage liver disease secondary to HCV infection, HCV envelope quasispecies were determined from intraoperative serum samples obtained before the anhepatic phase of transplantation and from liver biopsies 1.5 to 2.5 hours after the transplanted liver was perfused. Explanted (native) liver biopsies were taken as a control. Sequence analysis was performed on clones of specific HCV reverse transcriptase-polymerase chain reaction products spanning HVR1 and HVR2 of the E2 protein. HVR1 was more variable than HVR2 for all samples. Quasispecies isolated from postperfusion liver differed more from serum than did explanted liver quasispecies at HVR1 (P = 0.03) but not at HVR2 (P = 0.2). Comparison of HVR1 sequences from postperfusion liver versus serum revealed significantly less HVR1 genetic complexity and diversity (P = 0.02 and P = 0.04, respectively). Immediately after transplantation but before actual infection, liver allografts select out from the infecting serum inoculum a less heterogeneous, more closely related population of quasispecies variants.

Topics: Amino Acid Sequence; Blood Cells; Consensus Sequence; Genetic Variation; Hepacivirus; Hepatitis C; Humans; Liver; Liver Failure; Liver Transplantation; Molecular Sequence Data; Postoperative Period; Time Factors; Transplantation, Homologous; Viral Envelope Proteins; Viral Proteins

The diagnosis of ongoing hepatitis C virus (HCV) infection involves the detection of specific antibodies and of HCV-RNA. We aimed to assess the relationship between these two parameters in a representative sample of a population at high risk for HCV infection.. Plasma and serum samples were respectively tested for HCV-RNA by a qualitative PCR (Cobas Amplicor HCV, Roche) and for HCV antibodies by a MEIA screening assay (AxSYM HCV 3.0, Abbott) and an immunoblot (Inno-LIA-III, Innogenetics).. Out of 888 samples assayed, 579 (65.2%) were positive for HCV-RNA, while anti-HCV antibodies were detected respectively in 802 sera by AxSYM (90.3%) and in 783 by LIA (706 positive and 77 indeterminate, 88.2%). The anti-core antibodies displayed the best correlation with viremia, since they were present in 97.1% of the PCR+ samples, followed by anti-NS3 (90.2%) and anti-NS4 (89.6%). Only one HCV-RNA positive sample was negative by LIA and MEIA (early seroconversion). The AxSYM sample/cutoff (S/CO) values were directly correlated with the presence of HCV-RNA: a PCR positivity was found in 4.9% of samples with a S/CO < or =10, in 60.8% of samples with a S/CO between 11 and 50 and in 93.6% of cases with a S/CO >50, (P < 0.005). The immunoblot adds little, on a single specimen, to the information yielded by the AxSYM screening test. A suitable diagnostic algorithm for HCV in high-risk settings could be the anti-HCV screening by MEIA and a qualitative assay for HCV-RNA on samples with low reactivity.

Topics: Automation; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoblotting; Immunoenzyme Techniques; Reagent Kits, Diagnostic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins; Viremia

In the absence of satisfactory cell culture systems for hepatitis C virus (HCV), virtually all that is known about the proteins of the virus has been learned by the study of recombinant proteins. Characterization of virus proteins from patients with HCV has been retarded by the low virus titre in blood and limited availability of infected tissue. Here, the authors have identified a primary infection in a liver transplanted into an immunodeficient patient with chronic HCV. The patient required re-transplant and the infected liver, removed 6 weeks after the initial transplant, had a very high titre of HCV, 5 x 10(9) International Units (IU) per gram of liver. The density distribution of HCV in iodixanol gradients showed a peak at 1.04 g x ml(-1) with 73 % of virus below 1.08 g x ml(-1). Full-length HCV RNA was detected by Northern blotting and the ratio between positive- and negative-strand HCV RNA was determined as 60. HCV was partially purified by precipitation with heparin/Mn(2+) and a single species of each of the three structural proteins, core, E1 and E2, was detected by Western blotting. The molecular mass of core was 20 kDa, which corresponds to the mature form from recombinant sources. The molecular mass of glycoprotein E1 was 31 kDa before and 21 kDa after deglycosylation with PNGase F or endoglycosidase H. Glycoprotein E2 was 62 kDa before and 36 kDa after deglycosylation, but E2-P7 was not detected. This was in contrast to recombinant sources of E2 which contain E2-P7.

Topics: Base Sequence; Dimerization; Genome, Viral; Glycosylation; Hepacivirus; Hepatitis C; Humans; Liver; Molecular Sequence Data; RNA, Viral; Viral Envelope Proteins; Viral Structural Proteins

To investigate the immunogenicity of candidate DNA vaccine against hepatitis C virus (HCV) delivered by two plasmids expressing HCV envelope protein 1 (E1) and envelope protein 2 (E2) antigens respectively and to study the effect of CpG adjuvant on this candidate vaccine.. Recombinant plasmids expressing HCV E1 and E2 antigens respectively were used to simultaneously inoculate mice with or without CpG adjuvant. Antisera were then collected and titers of anti-HCV antibodies were analyzed by ELISA. One month after the last injection, animals were sacrificed to prepare single-cell suspension of splenocytes. These cells were subjected to HCV antigen specific proliferation assays and cytokine secretion assays to evaluate the cellular immune responses of the vaccinated animals.. Antibody responses to HCV E1 and E2 antigens were detected in vaccinated animals. Animals receiving CpG adjuvant had slightly lower titers of anti-HCV antibodies in the sera, while the splenocytes from these animals showed higher HCV-antigen specific proliferation. Analysis of cytokine secretion from the splenocytes was consistent with the above results. While no antigen-specific IL-4 secretion was detected for all vaccinated animals, HCV antigen-specific INF-gamma secretion was detected for the splenocytes of vaccinated animals. CpG adjuvant enhanced the secretion of INF-gamma but did not change the profile of IL-4 secretion.. Vaccination of mice with plasmids encoding HCV E1 and E2 antigens induces humoral and cellular immune responses. CpG adjuvant significantly enhances the cellular immune response.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Antibody Formation; Cell Division; Cell Line; CpG Islands; Cricetinae; Cytokines; Female; Hepatitis C; Hepatitis C Antigens; Immunity, Cellular; Kidney; Mice; Mice, Inbred BALB C; Oligodeoxyribonucleotides; Plasmids; Spleen; Vaccines, DNA; Viral Envelope Proteins; Viral Hepatitis Vaccines

As the human tetraspanin CD81 binds hepatitis C virus (HCV) envelope glycoprotein E2, we addressed the role CD81 may play in cellular trafficking of HCV envelope proteins. Studies on HCV life cycle are complicated by the lack of a robust cell culture system; we therefore transfected mammalian cells with HCV E1-E2 cDNA, with or without human CD81 (huCD81) cDNA. In the absence of huCD81, HCV envelope proteins are almost completely retained in the endoplasmic reticulum. Instead, when huCD81 is present, a fraction of HCV envelope proteins passes through the Golgi apparatus, matures acquiring complex sugars and is found extracellularly associated with exosomes. These are 60-100-nm membrane vesicles enriched in tetraspanins, released into the extracellular milieu by many cell types and having fusogenic activity. We also report that human plasma contains exosomes and that in HCV patients, viral RNA is associated with these circulating vesicles. We propose that the HCV-CD81 complex leaves cells in the form of exosomes, circulates in this form and exploits the fusogenic capabilities of these vesicles to infect cells even in the presence of neutralizing antibodies.

Topics: Animals; Antigens, CD; CHO Cells; Cricetinae; Cricetulus; Flow Cytometry; Hepacivirus; Hepatitis C; Humans; Immunoprecipitation; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Secretory Vesicles; Tetraspanin 28; Transfection; Viral Envelope Proteins

We initially evaluated in mice the ability of naked DNA encoding intracellular forms of the E1E2 envelope proteins from HCV to induce antibody responses and compared the responses induced with the same plasmid adsorbed onto cationic poly (lactide co-glycolide) (PLG) microparticles. Although naked DNA was only able to induce detectable responses at the 100 microg dose level, making this approach impractical for evaluation in larger animals, PLG/DNA induced detectable responses at 10 microg. In addition, the PLG/DNA microparticles induced significantly enhanced responses to naked DNA when compared at the same dose level. Remarkably, PLG/DNA induced comparable responses to recombinant E1E2 protein adjuvanted with the emulsion MF59. Furthermore, PLG/DNA effectively primed for a booster response with protein immunization, while naked DNA did not. Therefore, PLG/DNA was selected for further evaluation in a non-human primate model. In a study in rhesus macaques, PLG/DNA induced seroconversion in 3/3 animals following three immunizations. Although the antibody responses appeared lower than those induced with recombinant protein adjuvanted with MF59, following a fourth dose, PLG/DNA and protein induced comparable responses. However, a single booster dose of recombinant protein administered to the animals previously immunized with PLG/DNA induced much higher responses. In addition, one of three animals immunized with PLG/DNA showed a cytotoxic T lymphocyte response in peripheral blood lymphocytes. In conclusion, cationic PLG microparticles with adsorbed HCV DNA generates potent immune responses.

Topics: Adjuvants, Immunologic; Animals; Cations; Cytotoxicity Tests, Immunologic; DNA, Viral; Drug Carriers; Drug Delivery Systems; Hepatitis C; Hepatitis C Antibodies; Immunoglobulin G; Lactic Acid; Macaca mulatta; Mice; Microspheres; Plasmids; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Polysorbates; Squalene; Vaccines, DNA; Viral Envelope Proteins; Viral Hepatitis Vaccines

The study of hepatitis C virus (HCV), a major cause of chronic liver disease, has been hampered by the lack of a cell culture system supporting its replication. Here, we have successfully generated infectious pseudo-particles that were assembled by displaying unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles. The presence of a green fluorescent protein marker gene packaged within these HCV pseudo-particles allowed reliable and fast determination of infectivity mediated by the HCV glycoproteins. Primary hepatocytes as well as hepato-carcinoma cells were found to be the major targets of infection in vitro. High infectivity of the pseudo-particles required both E1 and E2 HCV glycoproteins, and was neutralized by sera from HCV-infected patients and by some anti-E2 monoclonal antibodies. In addition, these pseudo-particles allowed investigation of the role of putative HCV receptors. Although our results tend to confirm their involvement, they provide evidence that neither LDLr nor CD81 is sufficient to mediate HCV cell entry. Altogether, these studies indicate that these pseudo-particles may mimic the early infection steps of parental HCV and will be suitable for the development of much needed new antiviral therapies.

Topics: Animals; Antigens, CD; Cells, Cultured; Genes, Reporter; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Hepatocytes; Humans; Membrane Proteins; Neutralization Tests; Receptors, LDL; Recombinant Fusion Proteins; Tetraspanin 28; Tumor Cells, Cultured; Viral Envelope Proteins; Viral Structural Proteins; Virion; Virus Assembly

Chronic infection with hepatitis C virus (HCV) is associated with disturbances of B lymphocyte activation and function: autoantibody production, mixed cryoglobulinemia, and B cell lymphomas. It has been proposed that these abnormalities reflect chronic antigenic stimulation or aberrant signaling through the B cell coreceptor, the latter mediated by binding of the HCV E2 glycoprotein to CD81. To test this hypothesis, we measured expression of activation and differentiation markers on peripheral blood B cells from patients with chronic HCV infection. Thirty-six HCV patients with and without mixed cryoglobulinemia were compared with 18 healthy control volunteers and 17 sustained virologic responders who had cleared HCV infection. Ten of the 36 HCV patient samples showed increased B cell frequencies; B cell frequency was higher in patients with more severe hepatic fibrosis. However, these samples lacked evidence of Ag-driven activation or proliferation. The expanded cells were low in the activation markers CD25, CD69, CD71, CD80, and CD86. Proliferation of circulating B cells was unchanged in HCV patients. These cells did not express the differentiation marker CD27, suggesting that they were not enriched in memory B cells. Furthermore, the expanded B cells expressed both IgD and IgM, suggesting that they were antigenically naive. Together, these results indicate that B cell expansion in the peripheral blood of HCV patients is not associated with Ag-mediated activation and differentiation. Instead, factors other than antigenic stimulation may promote the accumulation of peripheral blood B cells with a naive phenotype in a subset of HCV patients.

Topics: Adolescent; Adult; Aged; Antigens, CD; B-Lymphocyte Subsets; CD5 Antigens; Cell Division; Female; Flow Cytometry; Hepatitis C; Humans; Immunophenotyping; Interphase; Ligands; Lymphocyte Activation; Lymphocyte Count; Male; Membrane Proteins; Middle Aged; Severity of Illness Index; Tetraspanin 28; Tumor Necrosis Factor Receptor Superfamily, Member 7; Viral Envelope Proteins

To determine the pattern and significance of the HCV genetic heterogeneity before and during treatment with recombinant-2b or lymphoblastoid alpha-interferon, hypervariable region 1 (HVR-1) and NS5A quasispecies were characterised by cloning and sequencing in 12 HCV-1b-infected subjects. Patients were either responder-relapsers or non-responders to treatment. Extensive amino acid sequence analysis was applied to reveal the significance of HCV variation at key sites within HVR-1 and NS5A regions. Genetic complexity, genetic diversity, and the non-synonymous to synonymous substitution ratios of HVR-1 quasispecies decreased during treatment in responder-relapser patients only, and more markedly so following lymphoblastoid alpha-interferon. In non-responders, the HVR-1 quasispecies broadened. Amino acids G406 and Q409, which represent a major viral epitope, were highly conserved throughout treatment. Responder-relapser patients had a higher mutation frequency in NS5A than non-responders. Lymphoblastoid alpha-interferon promoted the selection of intermediate Interferon Sensitivity Determining Region (ISDR) sequences, whereas recombinant-2b alpha-interferon favoured maintenance or selection of conserved ISDR sequences. Variability upstream of the ISDR was associated with treatment response, but the amino acid substitutions conferring higher replicative ability to in vitro HCV replicons were absent in in vivo isolates. In conclusion, the pattern of HVR-1 quasispecies evolution correlates with the clinical response, and the conservation of specific amino acids may be useful for immune targeting in vivo. In responder-relapser patients, the initial HVR-1 evolution resembles that found in sustained responders. Variability within the entire NS5A, as opposed to a single region (ISDR), may have a role in influencing alpha-interferon treatment outcome. A differential effect of different alpha-interferon preparations on HCV quasispecies kinetics may exist.

Topics: Amino Acid Sequence; DNA, Viral; Genetic Heterogeneity; Genetic Variation; Hepacivirus; Hepatitis C; Humans; Interferon-alpha; Molecular Sequence Data; Phylogeny; Viral Envelope Proteins; Viral Nonstructural Proteins

Hepatitis C virus (HCV) infects nearly 3% of the population of the world and is a major cause of liver disease. However, the mechanism whereby the virus targets the liver for infection remains unknown, because none of the putative cellular receptors for HCV are both expressed specifically in the liver and capable of binding HCV envelope glycoproteins. Liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) is a calcium-dependent lectin expressed on endothelial cells of liver and lymph nodes. Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a homologous molecule expressed on dendritic cells, binds HIV and promotes infection. By using a virus-binding assay, we demonstrate that L-SIGN and DC-SIGN specifically bind naturally occurring HCV present in the sera of infected individuals. Further studies demonstrate that binding is mediated by the HCV envelope glycoprotein E2 and is blocked by specific inhibitors, including mannan, calcium chelators, and Abs to the lectin domain of the SIGN molecules. Thus, L-SIGN represents a liver-specific receptor for HCV, and L-SIGN and DC-SIGN may play important roles in HCV infection and immunity.

Topics: Base Sequence; Binding Sites; Cell Adhesion Molecules; DNA, Complementary; DNA, Viral; HeLa Cells; Hepacivirus; Hepatitis C; Humans; In Vitro Techniques; Lectins, C-Type; Liver; Receptors, Cell Surface; Receptors, Virus; Recombinant Proteins; RNA, Viral; Viral Envelope Proteins

Hepatitis C virus (HCV) E2 protein was recently reported to have a double-stranded RNA-activated protein kinase-eukaryotic initiation factor 2alpha (PKR-eIF2alpha) phosphorylation homology domain (PePHD); PKR is induced by interferon (IFN). PePHD interacts with PKR and inactivates it. PePHD could be a predictor for IFN response, like the interferon sensitivity determination region (ISDR) of HCV NS5A. Several groups reported that PePHD is conserved, and mutations in this region do not correlate with IFN response. In this study, we further investigated the amino acid variation of PePHD among four major genotypes and its correlation with IFN response.. We enrolled 74 patients for this study and determined PePHD sequence of HCV derived from sera of patients infected with HCV genotype 1a (1 patient; nonresponder [NR]), 1b (36 patients; 4 complete responders [CR], 32 NR), 2a (29 patients; 17 CR, 12 NR), and 2b (8 patients; 3 CR, 5 NR). We also analyzed mutations in ISDR of HCV genotype 1b in 31 patients.. PePHD had several variations among four genotypes investigated. In patients infected with HCV genotype 1b, PePHD sequence was well conserved and seemed to have no correlation with IFN response. Mutations in ISDR were correlated with IFN response. In patients with HCV genotypes 2a and 2b, PePHD had multiple variations, and one particular motif, "RGQQ-" at the N-terminus, showed a close correlation with IFN resistance. All eight patients with HCV containing this motif were IFN nonresponders.. IFN resistance of HCV correlates with its "RGQQ-" motif at the N-terminus of PePHD in HCV genotype 2a and 2b. PePHD of HCV could be a predictor of IFN resistance in patients infected with HCV genotype 2a and 2b.

Topics: Adult; Aged; Amino Acid Sequence; Antiviral Agents; Conserved Sequence; Drug Resistance, Viral; eIF-2 Kinase; Eukaryotic Initiation Factor-2; Female; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Humans; Interferons; Japan; Male; Middle Aged; Mutation; Sequence Analysis, Protein; Sequence Homology, Amino Acid; Viral Envelope Proteins

To investigate sequence of the complete E region of genotype 2a hepatitis C virus genome and to set up the basis for the further study on biological functions of envelope proteins of HCV.. Two cDNA fragments of 770bp, 1,100bp long, from serum of a patients infected HCV, were amplified by reverse transcription nested polymerase chain reaction (RT-nPCR). After being digested with two restriction endonucleases, the two fragments were cloned into JM105 respectively, then sequenced.. The two corresponding nucleotide sequences were obtained and ligated. The complete nucleotide sequences of envelope region and corresponding deduced amino acid sequences were compared with those of HCV-1, HC-C2, HCV-BK, HC-J6 and HC-J8. The homology of nucleotide sequence of the isolated strains was 60.5%, 60.1%, 59.7%, 87.8% and 67.5% respectively; the amino acid sequence homology to the isolated strains was found to be 67?3%, 66.4%, 65.0%, 87.8%, 79.0%, respectively.. The results indicate that the isolated strain (HCV-JS) belongs to genotype 2a, but there is heterogeneity between HCV-JS and HC-J6 strain.

Topics: Amino Acid Sequence; Cloning, Molecular; DNA, Complementary; DNA, Viral; Genotype; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Viral Envelope Proteins; Viral Nonstructural Proteins

Monoclonal antibody D32.10 produced by immunizing mice with a hepatitis C virus (HCV)-enriched pellet obtained from plasmapheresis of a chronically HCV1b-infected patient binds HCV particles derived from serum of different HCV1a- and HCV1b-infected patients. Moreover, this monoclonal has been shown to recognize both HCV envelope proteins E1 and E2. In an attempt to provide novel insight into the membrane topology of HCV envelope glycoproteins E1 and E2, we localized the epitope recognized by D32.10 on the E1 and/or E2 sequence using Ph.D.-12 phage display peptide library technology. Mimotopes selected from the phage display dodecapeptide library by D32.10 shared partial similarities with 297RHWTTQGCNC306 of the HCV E1 glycoprotein and with both 613YRLWHYPCT621 and 480PDQRPYCWHYPPKPC494 of the HCV E2 glycoprotein. Immunoreactivity of D32.10 with overlapping peptides corresponding to these three HCV regions confirmed these localizations and suggested that the three regions identified are likely closely juxtaposed on the surface of serum-derived particles as predicted by the secondary model structure of HCV E2 derived from the tick-borne encephalitis virus E protein. This assertion was supported by the detection of specific antibodies directed against these three E1E2 regions in sera from HCV-infected patients.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Blotting, Western; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoenzyme Techniques; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Peptide Library; Protein Conformation; Viral Envelope Proteins; Viral Structural Proteins

We hypothesized that hepatocytes exposed to hepatitis C virus (HCV) and human immunodeficiency virus (HIV) might be injured via an "innocent bystander" mechanism due to cell-surface binding of viral proteins. To assess this, we studied the effects of HCV envelope protein E2 and T-tropic HIV envelope glycoprotein gp120 on hepatocytes and saw potent apoptosis. Either viral protein alone did not induce this effect. HCV E2 and M-tropic HIV gp120 also induced significant apoptosis. Blocking the CXCR4 receptor led to a reduction in apoptosis. HCV E2 and HIV gp120 acted collaboratively to trigger a specific set of downstream signaling events, including up-regulation of the Fas ligand and dephosphorylation of the anti-apoptotic molecule AKT. These results suggest that hepatic injury may occur in HCV/HIV coinfection through the induction of novel downstream signaling pathways and provide a rationale for therapeutic interventions that interfere with specific receptors and signaling molecules.

Topics: Apoptosis; Cell Line, Tumor; Fas Ligand Protein; Hepacivirus; Hepatitis C; Hepatocytes; HIV Envelope Protein gp120; HIV-1; Humans; In Situ Nick-End Labeling; Membrane Glycoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Transfection; Up-Regulation; Viral Envelope Proteins

Although DNA immunization is a safe and efficient method for inducing cellular immune responses, it generates relatively weak and slow immune responses. Here, we investigated the effect of hepatitis C virus (HCV) antigen modifications on the induction of T-cell responses in DNA immunization. It is likely that the strength of T-cell responses has an inverse relationship with the length of the insert DNA. Interestingly, a mixture of several plasmids carrying each gene induced a higher level of T-cell responses than a single plasmid expressing a long polyprotein. Moreover, the presence of a transmembrane domain in HCV E2 resulted in stronger T-cell responses against E2 protein than its absence. Taken together, our results indicate that the tailored modifications of DNA-encoded antigens are capable of optimizing the induction of T-cell responses which is required for eliminating the cells chronically infected with highly variable viruses such as HCV and human immunodeficiency virus.

Topics: Animals; Female; Genetic Engineering; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Immunization; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Plasmids; T-Lymphocytes; Vaccines, DNA; Viral Envelope Proteins; Viral Hepatitis Vaccines

DNA-based immunization may be of prophylactic and therapeutic value for hepatitis C virus (HCV) infection. In efforts to improve the immunogenicity of a plasmid expressing the second envelope protein (E2) of HCV, we evaluated in mice the role of the antigen localization and demonstrated that membrane-bound and secreted forms induced higher titers of E2-specific antibodies, as well as earlier and higher seroconversion rates, than the intracellular form, but all three forms induced strong CTL. We also investigated whether E2-specific antibody responses could be enhanced by CpG optimization of the plasmid backbone and showed that removal of neutralizing CpG dinucleotides did not have a significant effect but addition of 64 immunostimulatory CpG motifs significantly enhanced anti-E2 titers. These results may have implications for the design and development of HCV DNA vaccines.

Topics: Animals; Base Sequence; CpG Islands; DNA, Viral; Female; Genetic Vectors; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Mice; Mice, Inbred BALB C; T-Lymphocytes, Cytotoxic; Vaccines, DNA; Viral Envelope Proteins; Viral Hepatitis Vaccines

Topics: Hepatitis C; Hepatitis C Antibodies; Humans; Serologic Tests; Viral Envelope Proteins

We identified 15 patients with acute hepatitis C (AHC) among 29 healthy volunteers participating in 2 consecutive pharmacokinetics studies. Molecular techniques were used to determine the relatedness of viral strains, whereas clinical and virologic follow-up was started to establish the course and outcome of the acute infection. After presentation, serum liver enzymes and HCV RNA were monitored weekly for 4 months, then monthly for at least 12 months. Liver biopsy was performed 6 to 12 months after AHC diagnosis. Phylogenetic analysis of coding regions for the envelope glycoproteins E1 and E2 was performed. At presentation, all 15 patients tested HCV RNA-positive and had HCV genotype 2c. Phylogenetic analysis indicated a common source of infection. Fourteen patients agreed to be followed prospectively. Infection resolved spontaneously in 8 patients, HCV RNA becoming undetectable by 4 to 5 months after the presumed time of infection in 5 of them and by 8, 13, and 24 months in the remaining 3. Six patients developed chronic infection. Liver biopsies performed in 9 subjects who were HCV RNA-positive 6 months after AHC diagnosis revealed that the prevalent histologic finding was lobular inflammation. In conclusion, our homogeneous cohort showed a wide spectrum of clinical, virologic and histologic features, and, more importantly, short-term outcome differed noticeably despite the common source of infection.

Topics: Acute Disease; Adult; Disease Outbreaks; DNA, Viral; Female; Follow-Up Studies; Health Personnel; Hepacivirus; Hepatitis C; Humans; Liver; Male; Middle Aged; Pharmacokinetics; Phylogeny; RNA, Viral; Viral Envelope Proteins

We analyzed sequences of hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in six chimpanzees, experimentally infected with a single HCV inoculum, to clarify the correlation between HVR1 mutation and antibodies to HVR1. Two chimpanzees had been immunized with synthetic HVR1 peptides before HCV inoculation. All six animals became infected with HCV but cleared the infection within the acute phase. The major HVR1 sequences in longitudinal sera were unchanged in animals both with and without anti-HVR1 antibodies. Additionally, sequences of HVR1 variants in each chimpanzee converged after 11 to 19 weeks. The data show that anti-HVR1 antibodies are unlikely to drive variation in HVR1.

Topics: Acute Disease; Amino Acid Sequence; Animals; Hepatitis C; Hepatitis C Antibodies; Molecular Sequence Data; Pan troglodytes; RNA, Viral; Viral Envelope Proteins

Hepatitis C virus (HCV) is difficult to study due to the lack of an efficient cell culture system or small animal model. As a result, HCV-cell interactions are not well-defined. In addition, several studies have identified a subset of patients in whom HCV RNA is present, but HCV antibody is not detected. We produced recombinant baculoviruses that expressed HCV structural proteins (core, E1 and E2, nt 342-2651) or control proteins. The HCV structural protein precursor was processed into immunoreactive proteins of appropriate size, and sucrose density sedimentation and electron microscopy of infected cell lysates demonstrated particle formation. To evaluate HCV antigenicity, particularly in patients who tested negative for HCV antibody in commercial HCV immunoassays but had persistent viremia, we evaluated the virus-like particles (VLPs) in solid-phase immunoassays. VLPs reacted with sera from HCV antibody positive subjects in these solid phase immunoassays, but not with control sera. Plasma samples from 19% (5/26) of HCV antibody negative subjects who were persistently HCV RNA positive also reacted with the HCV VLPs. When incubated with MOLT-4 cells at 4 degrees C, HCV VLPs demonstrated cell binding, and behaved similar to plasma-derived HCV preparations in a flow cytometry-based cell binding assay. These data suggest that recombinant HCV VLPs may allow identification of HCV antibody in patients, including some patients with persistent viremia and who are seronegative with current assays. In addition, HCV VLPs seem useful for evaluating HCV-cell interactions.

Topics: Animals; Antigens, Viral; Baculoviridae; Gene Expression Regulation; Genetic Vectors; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoassay; Microscopy, Electron; Recombinant Proteins; Spodoptera; Viral Core Proteins; Viral Envelope Proteins; Viral Structural Proteins

In July 2000, symptomatic acute hepatitis C was diagnosed in five patients who had attended the emergency room of a municipal hospital on the same day, about 6 weeks before. Investigation of the remaining 65 patients visited at the emergency room on that day disclosed that 8 patients had a positive anti-hepatitis C virus (anti-HCV) test and 4 of them had biochemical evidence of acute anicteric hepatitis. HCV RNA was detected in 12 of the 13 anti-HCV-positive patients. Phylogenetic analysis of the nonstructural 5A (NS5A) and E2 regions showed that 10 patients, including all 9 with acute hepatitis, were infected with a closely related HCV strain, while the remaining 2 patients harbored unrelated strains. Flushing of intravenous catheters with heparin retrieved from a multidose heparin solution in saline was carried out for all the patients involved in the hepatitis outbreak but in only 1 of 23 (4%) matched controls recruited among HCV-noninfected patients attending the emergency room on the same day, and this was the only significant difference concerning risk factors for HCV infection between patients and controls. Thus, accidental contamination of a multidose heparin solution with blood from an unrecognized HCV carrier was identified as the source of this nosocomial outbreak of hepatitis C.

Topics: Adult; Aged; Aged, 80 and over; Cross Infection; Disease Outbreaks; Emergency Service, Hospital; Female; Hepacivirus; Hepatitis C; Hospitals, Urban; Humans; Male; Middle Aged; Molecular Sequence Data; RNA, Viral; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Nonstructural Proteins

We previously demonstrated that hepatitis C virus (HCV) binds to human CD81 through the E2 glycoprotein. Therefore, expression of the human CD81 molecule in transgenic mice was expected to provide a new tool to study HCV infection in vivo, as the chimpanzee is the only species currently available as a laboratory animal model that can be infected with HCV. We produced transgenic mice expressing the human CD81 protein in a wide variety of tissues. We confirmed binding of recombinant E2 glycoprotein to the liver tissue as well as to thymocytes and splenic lymphocytes in the transgenic mice. We inoculated chimpanzee plasma infected with HCV into these animals. None of these transgenic animals showed evidence of viral replication. Furthermore, human CD81 transgenic mice that lack expression of endogenous mouse CD81 were also resistant to HCV infection. We conclude that expression of human CD81 alone is insufficient to confer susceptibility to HCV infection in the mouse. The presence of additional possible factors for HCV infection is discussed.

Topics: Animals; Antigens, CD; Disease Susceptibility; Hepatitis C; Humans; Membrane Proteins; Mice; Mice, Transgenic; Tetraspanin 28; Viral Envelope Proteins; Virus Replication

Hepatitis C virus (HCV) exists as a complex swarm of genetically related viruses known as a quasispecies. Recent work has shown that quasispecies complexity and evolutionary rates are associated with the outcome of acute infection. Knowledge of how the virus population evolves at different stages of chronic infection is less clear. We have studied rates of evolution of the first hypervariable region (HVR1) of the E2 envelope protein in six individuals with disparate liver disease severity. These data show that virus populations present in individuals with mild non-progressive liver disease evolve in a typical Darwinian fashion, with a consistent accumulation of non-synonymous (amino acid-changing) substitutions. By contrast, the virus population remains relatively static in individuals with severe progressive liver disease. Possible mechanisms for this disparity are discussed.

Topics: Alanine Transaminase; Amino Acids; Base Sequence; Binding Sites; Evolution, Molecular; Genetic Variation; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Liver; Liver Diseases; Molecular Sequence Data; Mutagenesis; Nucleotides; Phylogeny; Protein Sorting Signals; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Load

Identify hepatitis C virus (HCV) sequences in E1 and E2 protein binding to HepG2.. Synthetic 20-mer long, ten-residue overlapped peptides, from E1 and E2 proteins, were tested in HepG2 or Raji cell-binding assays. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay for high activity binding peptides (HABPs). Receptors for HepG2 cell were determined by cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.. Twelve HABPs were found in HCV genotype 1a, allowing six hepatocyte-binding sequences (HBSs) to be defined: two peptide-binding regions in E1 HABPs 4913 (YQVRNSTGLYHVTNDCPNSS) and 4918 (MTPTVATRDGKLPATQLRRHY). Four hepatocyte-binding regions were defined in E2: region-I, peptide 4931 (ETHVTGGSAGHTVSGFVSLLY); region-II, 4937-4939 (HHKFNSSGCPERLASCRPLTDFDQGWGPISYANGSGPDQR); region-III, 4943-4945 (PVYCFTPSPVVVGTTDRSGAPTYSWGENDTDVFVLNNTR) and region-IV, 4949-4952 (CGAPPCVIGGAGNNTLHCPTDCFRKHPDATYSRCGSGPWITPRCLVDYPY). The underlined sequences are most relevant in the binding process. HABPs 4913 and 4938 also bind to CD81 positive Raji cells. Region-II 4938 HABPs bind to 50 and 60kDa HepG2 cell membrane surface proteins.. Six HVRs to the HepG2 were identified. Some HABPs have been previously found to be antigenic and immunogenic. HABPs, 4918 (from E1), 4938, 4949, 4950, 4951 and 4952 (from E2) have not been previously recognised. These HABPs could be relevant to HCV invasion of hepatocytes.

Topics: Amino Acid Sequence; Genotype; Hepacivirus; Hepatitis C; Humans; Iodine Radioisotopes; Liver Neoplasms; Molecular Sequence Data; Protein Binding; Tumor Cells, Cultured; Viral Envelope Proteins; Viral Proteins

We previously reported a number of features of hepatitis C virus (HCV) chimeric glycoproteins related to pseudotype virus entry into mammalian cells. In this study, pseudotype virus was neutralized by HCV E2 glycoprotein-specific antibodies and infected human sera. Neutralization (50% reduction of pseudotype virus plaque formation) was observed with two human immunoglobulin G1 monoclonal antibodies (MAbs) at concentrations of between 2.5 and 10 microg/ml. A hyperimmune rabbit antiserum to an E2 hypervariable region 1 (HVR1) mimotope also exhibited an HCV E2 pseudotype virus neutralization titer of approximately 1/50. An E1 pseudotype virus used as a negative control was not neutralized to a significant level (<1/10) by these MAbs or rabbit antiserum to E2 HVR1. Since HCV probably has a lipid envelope, the role of complement in antibody-mediated virus neutralization was examined. Significant increases in the neutralization titers of the human MAbs (approximately 60- to 160-fold higher) and rabbit antiserum to HVR1 mimotopes (approximately 10-fold higher) were observed upon addition of guinea pig complement. Further, these studies suggested that complement activation occurred primarily by the classical pathway, since a deficiency in the C4 component led to a significant decrease in the level of virus neutralization. This same decrease was not observed with factor B-deficient complement. We also determined that 9 of 56 HCV-infected patient sera (16%) had detectable pseudotype virus neutralization activity at serum dilutions of between 1/20 and 1/50 and that complement addition enhanced the neutralization activity of some of the HCV-infected human sera. Taken together, these results suggest that during infection, HCV E2 glycoprotein induces a weak neutralizing antibody response, that those antibodies can be measured in vitro by the surrogate pseudotype virus plaque reduction assay, and that neutralization function can be augmented by complement.

Topics: Antibodies, Monoclonal; Antibody-Dependent Enhancement; Complement System Proteins; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immune Sera; Neutralization Tests; Recombinant Fusion Proteins; Viral Envelope Proteins

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles, but at times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Topics: Animals; Cell Line; Cell Membrane; Cricetinae; Cytopathogenic Effect, Viral; Gene Expression; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Immunohistochemistry; Mammals; Protein Disulfide-Isomerases; Recombinant Fusion Proteins; Viral Core Proteins; Viral Envelope Proteins

A highly attenuated, recombinant rabies virus (RV) vaccine strain-based vector was utilized as a new immunization strategy to induce humoral and cellular responses against hepatitis C (HCV) glycoprotein E2. We showed previously that RV-based vectors are able to induce strong immune responses against human immunodeficiency virus type I (HIV-1) antigens. Here we constructed and characterized three replication-competent RV-based vectors expressing either both HCV envelope proteins E1 and E2 or a modified version of E2 which lacks 85 amino acids of its carboxy terminus and contains the human CD4 transmembrane domain and the CD4 or RV glycoprotein cytoplasmic domain. All three constructs stably expressed the respective protein(s) as indicated by Western blotting and immunostaining. Moreover, surface expression of HCV E2 resulted in efficient incorporation of the HCV envelope protein regardless of the presence of the RV G cytoplasmic domain, which was described previously as a requirement for incorporation of foreign glycoproteins into RV particles. Killed and purified RV virions containing HCV E2 were highly immunogenic in mice and also proved useful as a diagnostic tool, as indicated by a specific reaction with sera from HCV-infected patients. In addition, RV vaccine vehicles were able to induce cellular responses against HCV E2. These results further suggest that recombinant RVs are potentially useful vaccine vectors against important human viral diseases.

Topics: Animals; Female; Genetic Vectors; Hepacivirus; Hepatitis Antibodies; Hepatitis C; Immunization; Mice; Mice, Inbred BALB C; Rabies virus; Recombinant Proteins; T-Lymphocytes, Cytotoxic; Vaccines, Attenuated; Vaccines, Synthetic; Viral Envelope Proteins; Viral Hepatitis Vaccines

Infection with hepatitis C virus (HCV) generally progresses to chronic disease, although a minority of patients appear to clear viremia spontaneously. In this investigation, serum samples were analyzed for virological parameters, serum alanine aminotransferase (ALT) levels, and neutralizing antibody response against pseudotyped vesicular stomatitis virus (VSV) generated using chimeric envelope glycoprotein 1 (E1) or 2 (E2) of HCV. Testing of sequential serum samples that were collected beginning at the onset of acute-phase disease demonstrated intermittent viremia, elevated ALT levels, and detectable neutralization activity against VSV in 9 of 10 patients. Serum neutralization activity did not exhibit a correlation with the genotype of the infecting HCV or with virus load. On the other hand, patients with chronic HCV infection consistently had detectable amounts of virus present but no significant variation in ALT levels, and serum samples from a majority (>90%) of patients failed to show detectable neutralization activity.

Topics: Acute Disease; Adolescent; Adult; Alanine Transaminase; Chronic Disease; Female; Hepatitis C; Hepatitis C Antibodies; Humans; Male; RNA, Viral; Vesicular stomatitis Indiana virus; Viral Envelope Proteins

Forty-eight overlapping octapeptides covering highly conservative regions of E1 and E2 hepatitis C virus (HCV) envelope proteins were synthesized and tested by ELISA against different groups of sera obtained from HCV-infected patients. All sera from patients with acute infection, except a single case of serum reactivity with the region HINRTALN, were nonreactive with any peptide. Sera obtained from chronic patients reacted with 12 peptides from five selected regions. Two immunodominant B epitopes were found, one being the precisely mapped antigenic site RMAWDM positioned inside the earlier shown immunodominant epitope from E1, and the second site, PALSTGLIH from E2, detected for the first time. New minor antigenic site was determined as PTDCFRKH from E2. We found only minor seroreactivity for one of the putative sites involved in CD81 binding, PYCWHYAP.

Topics: Adolescent; Adult; Amino Acid Sequence; B-Lymphocytes; Binding, Competitive; Child, Preschool; Conserved Sequence; Epitope Mapping; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Middle Aged; Molecular Sequence Data; Peptides; Viral Envelope Proteins

Hepatitis C virus (HCV)-associated B cell lymphomas were previously shown to express a restricted repertoire of immunoglobulin V(H) and V(L) genes, V(H)1-69 and VkappaA27, respectively. Although this suggests a role for antigen selection in the pathogenesis of these lymphomas, the driving antigen involved in the clonal expansion has not been identified. B cell response to a viral antigen, the HCV envelope glycoprotein 2 (E2), was analyzed in an asymptomatic HCV-infected patient. Single B cells, immortalized as hybridomas and selected for binding E2, were analyzed for their V gene usage. Sequences of these V region genes demonstrated that each hybridoma expressed unique V(H) and V(L) genes. Remarkably, these anti-E2 hybridomas preferentially used the V(H)1-69 gene. Analysis of replacement to silent mutation ratios indicated that the genes underwent somatic mutation and antigenic selection. In a separate report, human anti-E2 antibodies were also shown to express the same V(H) gene. These data strengthen the hypothesis that the HCV-associated lymphomas are derived from clonally expanded B cells stimulated by HCV.

Topics: Amino Acid Sequence; Clonal Deletion; DNA Mutational Analysis; Genes, Immunoglobulin; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Hybridomas; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Lymphoma, B-Cell; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Sequence Homology, Amino Acid; Viral Envelope Proteins

We have analyzed three cases of hepatitis C virus (HCV)-infected recipients who received blood from HCV-infected donors. Two recipients were exposed to two different HCV RNA-positive donors, and one was exposed to a single donor. All parental genomes from the actual infecting units of blood and the recipients were defined, and their presence in the follow-up serum samples was determined using sensitive strain-specific assays. The strain from one of the donors was found to predominate in all recipients' serum samples collected throughout the follow-up period of 10 to 30 months. In two recipients exposed to two infected donors, the strain from the second donor was occasionally found at very low level. However, the original recipients' strains were not detected. Our observations show that HCV-infected individuals can be superinfected with different strains, and this event may lead to eradication or suppression of the original infecting strain. Furthermore, our findings demonstrate that simultaneous exposure to multiple HCV strains may result in concomitant infection by more than one strain, although a single strain could rapidly establish its dominance. The results of the present study suggest the existence of competition among infecting HCV strains which determines the ultimate outcome of multiple HCV exposure.

Topics: Adult; Base Sequence; Blood Donors; Female; Hepacivirus; Hepatitis C; Humans; Male; Middle Aged; Molecular Sequence Data; Polymorphism, Single-Stranded Conformational; RNA, Viral; Superinfection; Transfusion Reaction; Viral Envelope Proteins; Viral Nonstructural Proteins

Chronic hepatitis C virus (HCV) infection frequently develops into liver disease and is accompanied by extra-hepatic autoimmune manifestations. The tetraspanin CD81 is a putative HCV receptor as it binds the E2 envelope glycoprotein of HCV and bona fide HCV particles. Here we show that HCV E2 binding to CD81 on human cells in vitro lowers the threshold for IL-2 receptor alpha expression and IL-2 production, resulting in strongly increased T cell proliferation. HCV E2-induced co-stimulation also enhances the production of IFN-gamma and IL-4 and causes increased TCR down-regulation. This suggests that binding of HCV particles to CD81 on T cells in vivo may lead to activation by otherwise suboptimal stimuli. Therefore, co-stimulation of autoreactive T cells by HCV may contribute to liver damage and autoimmune phenomena observed in HCV infection.

Topics: Antigens, CD; CD28 Antigens; CD3 Complex; Cytokines; Hepatitis C; Humans; Interleukin-2; Lymphocyte Activation; Membrane Proteins; Receptors, Antigen, T-Cell; T-Lymphocytes; Tetraspanin 28; Viral Envelope Proteins

Two infants vertically infected with hepatitis C virus (HCV) were followed-up from 3 or 4 months to 2.5 years of age. We analyzed five complementary DNA (cDNA) clones from each patient and compared the genetic drift of the HCV E2 gene hypervariable region (HVR) between the two infants and between the infants and their mothers within the two families. The HCV strains initially detected in infant 1 were identical to those found in her mother, while the HCV strains initially detected in infant 2 were very different from those of her mother. The mutation rate of HVR-1 proteins was higher in mother 1 than in mother 2, but was 1.6 to 2-fold higher in infant 2 than in infant 1 during the follow-up period. Serum ALT levels or serum HCV-core protein activity did not correlate with the mutation rates of HVR-1 proteins in either infant. However, the mutation rate of HVR-1 proteins significantly increased from 6 months of age in both infants, with concomitantly increased serum HCV antibody (anti-HCV) levels.

Topics: Adult; Amino Acid Sequence; Female; Follow-Up Studies; Hepatitis C; Hepatitis C Antibodies; Humans; Immune System; Infant; Infectious Disease Transmission, Vertical; Male; Molecular Sequence Data; Mothers; Mutation; Time Factors; Viral Envelope Proteins; Viral Proteins

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.

Topics: Amino Acid Sequence; Conserved Sequence; Hepacivirus; Hepatitis C; Humans; Interferon-gamma; Molecular Sequence Data; Protein Conformation; Sequence Analysis, DNA; Structure-Activity Relationship; Viral Envelope Proteins; Viral Proteins

The antibody profile to various proteins of hepatitis C virus (HCV) was studied in 113 patients positive for HCV RNA in various disease statuses of hepatitis C (HC). A single peptide (E2/NS1, aa 413-436 of HCV polyprotein) chosen from a conserved region at the C-terminus of the hypervariable region (HVR) HVR1 of HCV was found to be sufficient for reliable diagnosis of the infection, even in the acute phase. Six hundred and one suspected HC cases and 200 voluntary blood donors were tested by this peptide. The sensitivity of detection of HCV antibodies by this peptide did not increase with addition of peptides from other HCV proteins. Our results clearly demonstrate that antibodies to HCV envelope proteins occur in a higher percentage of the infected population than those to other proteins. This emphasizes the necessity of using representative sequences from HCV envelope proteins in diagnostic immunoassays of this viral infection.

Topics: Acute Disease; Amino Acid Sequence; Case-Control Studies; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunoenzyme Techniques; Molecular Sequence Data; Sequence Homology, Amino Acid; Viral Envelope Proteins

The genome of hepatitis C virus (HCV) encodes two envelope glycoproteins (E1 and E2), which are thought to be responsible for receptor binding and membrane fusion resulting in virus penetration. To investigate cell surface determinants important for HCV infection, we used a recombinant vesicular stomatitis virus (VSV) in which the glycoprotein gene was replaced with a reporter gene encoding green fluorescent protein (GFP) and produced HCV-VSV pseudotypes possessing chimeric HCV E1 or E2 glycoproteins, either individually or together. The infectivity of the pseudotypes was determined by quantifying the number of cells expressing the GFP reporter gene. Pseudotypes that contained both of the chimeric E1 and E2 proteins exhibited 10--20 times higher infectivity on HepG2 cells than the viruses possessing either of the glycoproteins individually. These results indicated that both E1 and E2 envelope proteins are required for maximal infection by HCV. The infectivity of the pseudotype virus was not neutralized by anti-VSV polyclonal antibodies. Bovine lactoferrin specifically inhibited the infection of the pseudotype virus. Treatment of HepG2 cells with Pronase, heparinase, and heparitinase but not with phospholipase C and sodium periodate reduced the infectivity. Therefore, cell surface proteins and some glycosaminoglycans play an important role in binding or entry of HCV into susceptible cells. The pseudotype VSV possessing the chimeric HCV glycoproteins might offer an efficient tool for future research on cellular receptors for HCV and for the development of prophylactics and therapeutics for hepatitis C.

Topics: Animals; Cell Line; CHO Cells; Cricetinae; Hepacivirus; Hepatitis C; Humans; Recombinant Fusion Proteins; Vesicular stomatitis Indiana virus; Viral Envelope Proteins; Viral Structural Proteins

The genotype of hepatitis C virus (HCV) can profoundly affect the success of antiviral therapy for HCV infection. A possible contributing factor is a varied immune response elicited by infection with different HCV genotypes. In this study, full-length E2 proteins of HCV genotypes 1a, 1b, 2a, and 2b were used to determine the fraction of the humoral immune response to HCV E2 that is genotype specific. Greater than 90% of all infected individuals had serum antibodies to the four E2 proteins. Overall, individuals infected with genotype 1a or 1b were characterized by variable immune responses to HCV E2 with relatively high amounts of cross-reactivity with other E2 proteins. Individuals infected with genotype 2a or 2b exhibited a strong preferential reactivity to genotype 2a and 2b E2 proteins. Individuals with elevated titers to HCV E2 were more likely to be infected with genotype 2a and had a significantly lower median viral load. These findings indicate that the antibody response to HCV E2 is affected by the genotype of the virus and that induction of a strong humoral immune response to HCV E2 may contribute to a decreased viral load.

Topics: Adult; Aged; Cross Reactions; Female; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Middle Aged; Viral Envelope Proteins; Viral Load

Clinical and experimental evidence indicates that the hepatitis C virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate for the development of an effective anti-HCV vaccine. Identification of the human epitopes that are conserved among isolates and are able to elicit protective antibodies would constitute a significant step forward. This work describes the mapping of the B-cell epitopes present on the surface of HCV/E2, as recognized by the immune system during infection, by the analysis of the reciprocal interactions of a panel of human recombinant Fabs derived from an HCV-infected patient. Three unrelated epitopes recognized by antibodies with no neutralization-of-binding (NOB) activity were identified; a fourth, major epitope was defined as a clustering of minor epitopes recognized by Fabs endowed with strong NOB activity.

Topics: Antibodies, Monoclonal; B-Lymphocytes; Epitope Mapping; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Humans; Peptide Library; Viral Envelope Proteins

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is a highly heterogeneous sequence that is promiscuously recognized by human sera via binding to amino acid residues with conserved physicochemical properties. We generated a panel of mAbs from mice immunized with HVR1 surrogate peptides (mimotopes) affinity-selected with sera from HCV-infected patients from a phage display library. A high number of specific clones was obtained after immunization with a pool of nine mimotopes, and the resulting mAbs were shown to recognize several 16- and 27-mer peptides derived from natural HVR1 sequences isolated from patients with acute and chronic HCV infection, suggesting that HVR1 mimotopes were efficient antigenic and immunogenic mimics of naturally occurring HCV variants. Moreover, most mAbs were shown to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded HVR1. In addition, a highly promiscuous mAb was able to specifically capture bona fide viral particles (circulating HCV RNA) as well as rHCV-like particles assembled in insect cells expressing structural viral polypeptides derived from an HCV 1a isolate. These findings demonstrate that it is possible to induce a broadly cross-reactive clonal Ab response to multiple HCV variants. In consideration of the potentially important role of HVR1 in virus binding to cellular receptor(s), such a mechanism could be exploited for induction of neutralizing Abs specific for a large repertoire of viral variants.

Topics: Antibodies, Monoclonal; Antibodies, Viral; Antibody Specificity; Antigens, Viral; Cross Reactions; Epitope Mapping; Hepacivirus; Hepatitis C; Humans; Peptides; Protein Folding; Viral Envelope Proteins; Virion

The envelope glycoprotein E2 of hepatitis C virus (HCV) has been shown to bind human target cells. Anti-E2 antibodies have been associated with both recovery from natural infection in humans and protection from challenge with homologous HCV in chimpanzees. Therefore E2 has become a major target for the development of anti-HCV vaccines. Two E2 fragments [amino acids (aa) 450-565 and aa 385-565] derived from a subtype 1b HCV genome were expressed as N-terminally hexahistidine-tagged proteins in Escherichia coli and purified to over 85% purity. Both proteins were specifically recognized by homologous hepatitis-C-patient's serum on Western blotting, suggesting that these E. coli-derived E2 proteins displayed E2-specific antigenicity. E2-116 (aa 450-565) elicited strong antibody responses in BALB/c mice and rabbits. Rabbit antiserum raised against renatured E2-116 (R(E2-116R)) was able to recognize subtype 1b and 1a E2 glycoproteins expressed in mammalian cells on Western blotting. E2-181 (aa 385-565) reacted with 40% of anti-HCV(+) patients' sera in ELISA. R(E2-116R) and E2-181 were successfully used in preliminary modified vaccinia virus Ankara- and DNA-based E2 vaccine studies for detecting antigen expression in vitro and assessing induced humoral immune responses in mice. The E2 proteins and rabbit antiserum reported here could find wider applications in the development of effective diagnostic, prophylactic and therapeutic measures against HCV.

Topics: Animals; Blotting, Western; Cell Line; CHO Cells; Cricetinae; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Female; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immune Sera; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Plasmids; Rabbits; Recombinant Fusion Proteins; Viral Envelope Proteins; Viral Hepatitis Vaccines

Chronic hepatitis C virus (HCV) infection has been associated with development of mixed cryoglobulinemia type 2 (MC2), a lymphoproliferative disorder characterized by B cell monoclonal expansion and immunoglobulin M/k cryoprecipitable immunoglobulin production. A short sequence (codons 384-410) of the HCV E2 protein, which has the potential to promote B cell proliferation, was investigated in 21 patients with HCV-related MC2 and in a control group of 20 HCV carriers without MC2. In 6 of the 21 (29%) patients with MC2, all the clones isolated from plasma, peripheral blood mononuclear cells, and liver showed sequence length variation compared with the hypervariable region 1 (HVR1) consensus sequence; 5 patients had an insertion at codon 385, and 1 patient had a deletion at codon 384. Inserted residues at position 385 were different within and between patients. No such mutations were observed in any of the HVR1 clones from control patients without MC2, and the difference between the 2 groups was statistically significant (P =.02). Analysis of 1345 HVR1 sequences obtained from GenBank strongly supported the conclusion that the observed insertions and deletion represent a rare event in HCV-infected patients, suggesting that they are significantly associated with MC2. The physical and chemical profiles of the 385 inserted residues detected in the MC2 patients were consistent with the possibility that these mutations, which occurred in a region containing immunodominant epitopes for neutralizing antibodies and binding sites for B lymphocytes, may be selected by functional constraints for interaction with host cells.

Topics: Adult; Aged; Amino Acid Sequence; Base Sequence; Cloning, Molecular; Cryoglobulinemia; DNA Transposable Elements; Hepatitis C; Humans; Infant; Leukocytes, Mononuclear; Liver; Male; Middle Aged; Molecular Sequence Data; Phylogeny; RNA, Viral; Sequence Alignment; Viral Envelope Proteins; Viral Proteins

Several hepatitis C virus (HCV) proteins have been shown in vitro to interact with host cellular components that are involved in immune regulation. However, there is a paucity of data supporting the relevance of these observations to the in vivo situation. To test the hypothesis that such an interaction suppresses immune responses, we studied a line of transgenic C57BL/6 mice that express the HCV core and envelope proteins in the liver. The potential effects of these proteins on the hepatic immune response were evaluated by challenging these mice with a hepatotropic adenovirus. Both transgenic and nontransgenic mice developed similar courses of infection and cleared the virus from the liver by 28 days postinfection. Both groups of mice mounted similar immunoglobulin G (IgG), IgG2a, interleukin-2, and tumor necrosis factor alpha responses against the virus. Additionally, BALB/c mice were able to clear infection with recombinant adenovirus that does or does not express the HCV core and envelope 1 proteins in the same manner. These data suggest that HCV core and envelope proteins do not inhibit the hepatic antiviral mechanisms in these murine experimental systems and thus favor a model in which HCV circumvents host responses through a mechanism that does not involve general suppression of intrahepatic immune responses.

Topics: Animals; Apoptosis; Hepatitis C; Immune Tolerance; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Tumor Necrosis Factor-alpha; Viral Core Proteins; Viral Envelope Proteins

Hepatitis C virus (HCV) infection is associated with extrahepatic B-cell lymphoproliferative disorders. To determine whether a viral antigen drives this B-cell expansion, the B-cell receptors were cloned from HCV-associated lymphomas and were expressed as soluble immunoglobulins. The rescued immunoglobulins were then tested for their ability to bind the HCV-E2 envelope glycoprotein, an antigen that was previously implicated in the pathogenesis of HCV-associated B-cell diseases. One of 2 lymphoma immunoglobulin test cases bound the E2 protein in a manner identical to a bona fide human anti-E2 antibody. Moreover, it bound E2 from multiple viral genotypes, suggesting reactivity with a conserved E2 epitope. These findings support the hypothesis that some HCV-associated lymphomas originate from B cells that were initially activated by the HCV-E2 protein and might explain the association between HCV infection and some B-cell lymphoproliferative disorders.

Topics: Amino Acid Sequence; B-Lymphocytes; Cloning, Molecular; Epitopes; Gene Expression; Genotype; Hepacivirus; Hepatitis C; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Immunoglobulins; Lymphoma, Non-Hodgkin; Molecular Sequence Data; Mutation; Protein Conformation; Receptors, Antigen, B-Cell; Viral Envelope Proteins

CD8(+) cytotoxic T lymphocytes (CTL) are thought to control hepatitis C virus (HCV) replication and so we investigated why this response fails in persistently infected individuals. The HCV quasispecies in three persistently infected chimpanzees acquired mutations in multiple epitopes that impaired class I MHC binding and/or CTL recognition. Most escape mutations appeared during acute infection and remained fixed in the quasispecies for years without further diversification. A statistically significant increase in the amino acid replacement rate was observed in epitopes versus adjacent regions of HCV proteins. In contrast, most epitopes were intact when hepatitis C resolved spontaneously. We conclude that CTL exert positive selection pressure against the HCV quasispecies and the outcome of infection is predicted by mutations in class I MHC restricted epitopes.

Topics: Acute Disease; Amino Acid Sequence; Animals; Antigenic Variation; Cell Line; Epitopes; Follow-Up Studies; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Hepatitis C, Chronic; Histocompatibility Antigens Class I; Molecular Sequence Data; Mutation; Pan troglodytes; Remission, Spontaneous; RNA, Viral; Sequence Alignment; Sequence Homology, Amino Acid; T-Lymphocytes, Cytotoxic; Viral Envelope Proteins; Viral Nonstructural Proteins

To find neutralizing antibody candidates against hepatitis C virus (HCV) infection.. We constructed two eukaryotic expression vectors which contained the E1 and E2 gene of HCV, and detected their expression in mammalian cells with transient expression. BALB/c mice were given subculaneous injections of constructed vectors combined with the IL-2 gene intraepidermally and evaluated for induced humoral immune responses by enzyme-linked immunosorbent assay (ELISA). We used an antibody-virus interaction assay to analyze the interaction of the antisera and HCV viral particles in vitro.. Anti E1 and anti-E2 antisera were obtained from immunized mice. The serum of mice immunized with the E2 gene immunoprecipitated the HCV isolate in source serum and reacted with the isolates unrelated to the original one.. Anti-E2 antibody in induced mice can cross-reactively capture HCV particles, highlighting the possibility of generating broadly reactive anti-E2 antibodies.

Topics: Animals; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Female; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Mice; Mice, Inbred BALB C; Viral Envelope Proteins

The quasispecies nature of the hepatitis C virus (HCV) is thought to play a central role in maintaining and modulating viral replication. Several studies have tried to unravel, through the parameters that characterize HCV circulating quasispecies, prognostic markers of the disease. In a previous work we demonstrated that the parameters of circulating viral quasispecies do not always reflect those of the intrahepatic virus. Here, we have analyzed paired serum and liver quasispecies from 39 genotype 1b-infected patients with different degrees of liver damage, ranging from minimal changes to cirrhosis. Viral level was quantified by real-time reverse transcription-PCR, and viral heterogeneity was characterized through the cloning and sequencing of 540 HCV variants of a genomic fragment encompassing the E2-NS2 junction. Although in 95% of patients, serum and liver consensus HCV amino acid sequences were identical, quasispecies complexity varied considerably between the viruses isolated from each compartment. Patients with HCV quasispecies in serum more complex (26%) than, less complex (28%) than, or similarly complex (41%) to those in liver were found. Among the last, a significant correlation between fibrosis and all the parameters that measure the viral amino acid complexity was found. Correlation between fibrosis and serum viral load was found as well (R = 0.7). With regard to the origin of the differences in quasispecies complexity between serum and liver populations, sequence analysis argued against extrahepatic replication as a quantitatively important contributing factor and supported the idea of a differential effect or different selective forces on the virus depending on whether it is circulating in serum or replicating in the liver.

Topics: Adult; Aged; Amino Acids; Base Sequence; DNA, Viral; Female; Hepacivirus; Hepatitis C; Humans; Liver; Male; Middle Aged; Molecular Sequence Data; Viral Envelope Proteins; Viral Nonstructural Proteins

Six donor-recipient clusters of hepatitis C virus (HCV)-infected individuals were studied. For five clusters the period of infection of the donor could be estimated, and for all six clusters the time of infection of the recipients from the donor via blood transfusion was also precisely known. Detailed phylogenetic analyses were carried out to investigate the genomic evolution of the viral quasispecies within infected individuals in each cluster. The molecular clock analysis showed that HCV quasispecies within a patient are evolving at the same rate and that donors that have been infected for longer time tend to have a lower evolutionary rate. Phylogenetic analysis based on the split decomposition method revealed different evolutionary patterns in different donor-recipient clusters. Reactivity of antibody against the first hypervariable region (HVR1) of HCV in donor and recipient sera was evaluated and correlated to the calculated evolutionary rate. Results indicate that anti-HVR1 reactivity was related more to the overall level of humoral immune response of the host than to the HVR1 sequence itself, suggesting that the particular sequence of the HVR1 peptides is not the determinant of reactivity. Moreover, no correlation was found between the evolutionary rate or the heterogeneity of the viral quasispecies in the patients and the strength of the immune response to HVR1 epitopes. Rather, the results seem to imply that genetic drift is less dependent on immune pressure than on the rate of evolution and that the genetic drift of HCV is independent of the host immune pressure.

Topics: Blood Donors; Cluster Analysis; Evolution, Molecular; Genotype; Hepacivirus; Hepatitis C; Humans; Peptides; Phylogeny; Transfusion Reaction; Viral Envelope Proteins

Topics: Amino Acid Sequence; Drug Resistance, Microbial; eIF-2 Kinase; Eukaryotic Initiation Factor-2; Genotype; Hepacivirus; Hepatitis C; Humans; Interferon-alpha; Phosphorylation; Viral Envelope Proteins

The mechanisms by which hepatitis C virus (HCV) induces chronic infection in the vast majority of infected individuals are unknown. Sequences within the HCV E1 and E2 envelope genes were analyzed during the acute phase of hepatitis C in 12 patients with different clinical outcomes. Acute resolving hepatitis was associated with relative evolutionary stasis of the heterogeneous viral population (quasispecies), whereas progressing hepatitis correlated with genetic evolution of HCV. Consistent with the hypothesis of selective pressure by the host immune system, the sequence changes occurred almost exclusively within the hypervariable region 1 of the E2 gene and were temporally correlated with antibody seroconversion. These data indicate that the evolutionary dynamics of the HCV quasispecies during the acute phase of hepatitis C predict whether the infection will resolve or become chronic.

Topics: Acute Disease; Adult; Aged; Antibodies, Viral; Disease Progression; Evolution, Molecular; Female; Genes, Viral; Genetic Variation; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Male; Middle Aged; Molecular Sequence Data; Phylogeny; Prospective Studies; Selection, Genetic; Time Factors; Viral Envelope Proteins; Virus Replication

Defined regions of hepatitis C virus (HCV) envelope 2 (E2), PePHD, and nonstructural 5A (NS5A) protein (PKR-binding domain) have been shown to interact with interferon alfa (IFN-alpha)-inducible double-stranded RNA-activated protein kinase (PKR) in vitro, suggesting a possible mechanism of HCV to evade antiviral effects of IFN-alpha. The clinical correlation between amino acid mutations within the E2 PePHD or the NS5A PKR-binding domain and response to antiviral treatment in HCV-3a-infected patients is unknown. Thirty-three patients infected with HCV-3a isolates were treated with IFN-alpha with or without ribavirin. The carboxyterminal half of E2 and of the NS5A gene were sequenced. Sixteen patients achieved a sustained virological response (SR), 6 patients an end-of-treatment response with relapse thereafter (ETR), and 11 patients were nonresponders (NR). Within the PePHD of the E2 protein 0.5 (range, 0-2) mutations were observed in SR patients, whereas the number of mutations in ETR or NR patients was 0.2 (0-1). Quasispecies analyses showed almost no heterogeneity. The mean number of mutations within the PKR-binding domain of the NS5A protein was 1.6 (range, 0-4) in SR patients, 1 (0-2) in ETR patients, and 1.6 (0-3) in NR patients. Patients with higher numbers of mutations within the E2 or NS5A region showed a trend towards lower pretreatment viremia. Phylogenetic and conformational analyses of E2 or NS5A sequences allowed no differentiation between sensitive and resistant isolates. However, mutations within the E2 PePHD in SR patients were frequent, and hydrophobic mutations within the hydrophilic area of PePHD at codon 668 and 669 were exclusively observed in sustained virological responders.

Topics: Adult; Amino Acid Sequence; Antiviral Agents; Drug Therapy, Combination; Female; Hepacivirus; Hepatitis C; Humans; Interferon-alpha; Male; Middle Aged; Molecular Conformation; Molecular Sequence Data; Mutation; Phylogeny; Ribavirin; Viral Envelope Proteins; Viral Nonstructural Proteins

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.

Topics: Animals; Antigens, CD; Drug Design; Endoplasmic Reticulum; Evaluation Studies as Topic; Female; Glycosylation; Guinea Pigs; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunization; Membrane Proteins; Mice; Mice, Inbred C57BL; Recombinant Proteins; Tetraspanin 28; Vaccines, DNA; Viral Envelope Proteins; Viral Hepatitis Vaccines

Hepatitis C virus (HCV) infection not only causes chronic liver diseases but shows extrahepatic manifestations as oral lichen planus (OLP) and oral cancer. To elucidate the direct relationships among these diseases and HCV infection, we investigated the detection of positive- and negative-strand HCV-RNA from serum, OLP (n=19), and oral cancer (n=17) tissues. We used a sensitive reverse transcription to polymerase chain reaction (RT-PCR) method, and analyzed sequences from the HCV El/E2 region of the genome from serum and tissue. Positive and negative HCV-RNA strands were observed in 13 (92.9%) and 3 (21.4%) OLP tissues, respectively. In oral cancer tissues, positive HCV-RNA strands were detected in all tissues from anti-HCV positive patients. Negative HCV-RNA strands were observed in 5 of 7 (71.4%) patient's tissues. Furthermore, it was confirmed that the sequence from one of each OLP and oral cancer patient differed between serum and tissue HCV-RNA. These results may indicate that HCV persists and replicates in these lesions, suggesting a pathological role for HCV, although the mechanisms are unclear.

Topics: Adult; Aged; Aged, 80 and over; DNA, Viral; Female; Genome, Viral; Hepacivirus; Hepatitis C; Humans; Lichen Planus, Oral; Male; Middle Aged; Mouth Neoplasms; Viral Envelope Proteins; Virus Replication

The hepatitis C virus (HCV) envelope glycoprotein-2 inhibits the interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) via the PKR eukaryotic initiation factor-2alpha phosphorylation homology domain (PePHD). The present study examined the genetic variability of the PePHD in patients receiving IFN therapy. The PePHD from 12 HCV genotype 1 (HCV-1)-infected patients receiving daily IFN therapy was amplified by reverse-transcriptase polymerase chain reaction and analyzed by direct and clonal sequencing. The PePHD was highly conserved in 38 HCV GenBank isolates. There was no difference in pretreatment PePHD sequences isolated from IFN responders versus nonresponders. The major PePHD quasi-species variant did not change after 6 weeks of daily IFN therapy, and in 1 patient the major quasi-species variant did not change during 9 months of observation. Sequencing of 25 pretreatment PePHD clones from 3 patients confirmed that there was extremely low sequence variability surrounding the PePHD. The PePHD is highly conserved in HCV-1-infected IFN responders and nonresponders and does not appear to evolve in response to IFN therapy.

Topics: Amino Acid Sequence; Binding Sites; Conserved Sequence; eIF-2 Kinase; Eukaryotic Initiation Factor-2; Genotype; Glycoproteins; Hepacivirus; Hepatitis C; Humans; Interferons; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Viral Envelope Proteins

The evolution of hepatitis C virus (HCV) envelope variation was studied using a liver-transplant model to evaluate the role of HCV quasispecies for hepatocyte infection. Twelve HCV polymerase chain reaction (PCR)-positive liver-transplant recipients (6 with posttransplantation biochemical hepatitis and 6 without hepatitis [controls]) were prospectively evaluated and underwent detailed quasispecies analysis of pre- and postoperative serum samples. HCV amino acid sequence diversity and complexity at the first hypervariable region (HVR1) of the second envelope protein (E2) was correlated with outcome. Recurrence of HCV-induced allograft injury was defined by persistently elevated serum alanine transaminase (ALT) levels. The major variant (sequences >10% of all clones) of recipients with hepatitis accounted for a significantly smaller percent of all preoperative clones compared with controls (41% +/- 6% vs. 69% +/- 8%; P <.015). Recipients with hepatitis had an increased number of pretransplantation major variants (2.5 +/- 0.3 vs. 1.1 +/- 0.2; P <.006). Eighty-three percent of controls had a predominant variant (accounting for >50% of clones) compared with 17% of those with recurrence (P <.05). These differences did not persist postoperatively. In addition, recipients without a pretransplantation predominant variant demonstrated an increased allograft fibrosis score (2.3 +/- 0.3 vs. 0.5 +/- 0.3; P <.015) at 181 to 360 days posttransplantation compared with those in whom a predominant variant was present. Increased HCV envelope complexity may act as a stronger antigenic stimulus or improve hepatocyte receptor binding and lead to allograft hepatitis and fibrosis. Although pretransplantation differences in HCV quasispecies did not persist postoperatively, pretransplantation quasispecies may be a predictor of HCV-induced hepatitis and graft fibrosis after liver transplantation.

Topics: Adult; Alanine Transaminase; Female; Hepacivirus; Hepatitis C; Humans; Liver Transplantation; Male; Middle Aged; Recurrence; Transplantation, Homologous; Viral Envelope Proteins

Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. Development of vaccines to prevent HCV infection, or at least prevent progression to chronicity, is a major goal. In mice and rhesus macaques, a DNA vaccine encoding cell-surface HCV-envelope 2 (E2) glycoprotein stimulated stronger immune responses than a vaccine encoding intracellular E2. Therefore, we used DNA encoding surface-expressed E2 to immunize chimpanzees 2768 and 3001. Chimpanzee 3001 developed anti-E2 after the second immunization and antibodies to hypervariable region 1 (HVR1) after the third immunization. Although chimpanzee 2768 had only low levels of anti-E2 after the third immunization, an anamnestic response occurred after HCV challenge. CTL responses to E2 were not detected before challenge, but a strong response was detected after HCV challenge in chimpanzee 2768. An E2-specific CD4+ response was detected in chimpanzee 2768 before challenge and in both chimpanzees postchallenge. Three weeks after the last immunization, animals were challenged with 100 50% chimpanzee-infectious doses (CID(50)) of homologous monoclonal HCV. As a control, a naive chimpanzee was inoculated with 3 CID(50) of the challenge virus. The vaccine did not generate sterilizing immunity because both vaccinated chimpanzees were infected. However, both vaccinated chimpanzees resolved the infection early whereas the control animal became chronically infected. Compared with the control animal, hepatitis appeared earlier in the course of the infection in both vaccinated chimpanzees. Therefore, DNA vaccine encoding cell surface-expressed E2 did not elicit sterilizing immunity in chimpanzees against challenge with a monoclonal homologous virus, but did appear to modify the infection and might have prevented progression to chronicity.

Topics: Animals; Antibodies, Monoclonal; CD4-Positive T-Lymphocytes; DNA; Female; Hepacivirus; Hepatitis C; Pan troglodytes; Plasmids; T-Lymphocytes, Cytotoxic; Vaccination; Viral Envelope Proteins

Topics: Acute Disease; Animals; Genetic Variation; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Mice; Mutation; Neutralization Tests; T-Lymphocytes, Cytotoxic; Viral Envelope Proteins

The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antibodies, Viral; Antigens, CD; Cell Line; Conserved Sequence; Epitope Mapping; Genotype; Hepacivirus; Hepatitis C; Humans; Membrane Proteins; Molecular Sequence Data; Protein Conformation; Recombinant Proteins; Tetraspanin 28; Viral Envelope Proteins

Changes in the deduced amino acid sequence of the envelope 2 (E2) region of the GB virus C/hepatitis G virus (GBV-C/HGV) were analyzed to investigate whether or not the region contributes to persistent infection with the virus.. Eight patients with acute hepatitis C and 1 patient with acute hepatitis of unknown etiology were included in the study. GBV-C/HGV RNA was detected in 6 patients, including the patient with hepatitis of unknown origin. The nucleotide sequence of the E2 region of hepatitis C virus (HCV) and GBV-C/HGV was determined by direct sequencing of polymerase chain reaction products in 5 patients with HCV infection and in 6 patients with GBV-C/HGV infection twice during the period of early infection and several months or years later in each patient.. The mean substitution rate of the deduced amino acid sequence in the E2 region was over 100 times lower (p < 0.001) in GBV-C/HGV (0.01 +/- 0.04/month/100 sites) than in HCV (2.4 +/- 1.7/month/100 sites). The amino acid sequence of the loop domain of GBV-C/HGV-E2 did not change in any of the 6 patients. On the other hand, the sequence of the hypervariable region of HCV-E2 changed remarkably (5.9 +/- 4.3/month/100 sites). No amino acid substitution in the loop domain was observed in 7 additional patients who showed persistent GBV-C/HGV viremia for more than 2 years.. These results indicate that changes in the amino acid sequence of the E2 region are not involved in the mechanism of persistent GBV-C/HGV infection.

Topics: Acute Disease; Adult; Amino Acid Sequence; Amino Acid Substitution; Female; Flaviviridae; Hepacivirus; Hepatitis C; Hepatitis, Viral, Human; Humans; Male; Middle Aged; Molecular Sequence Data; Sequence Alignment; Sequence Analysis, Protein; Viral Envelope Proteins

Both a double-stranded RNA-dependent protein kinase (PKR)-phosphorylation homology domain (PePHD) within the E2 protein and a PKR-binding domain within the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) genotype 1 isolates inhibit the function of the interferon alfa (IFN-alpha)-induced antiviral effector protein PKR in vitro. We investigated whether the mutational pattern of the E2 region (codons 618-681, including PePHD) of 81 HCV genotype 1-infected patients (HCV-1b [n = 54], HCV-1a [n = 27]) influences the response to IFN-alpha. Initial viral decline (DeltaHCV RNA) was determined at week 1 hereby covering the effector reactions of IFN-alpha-mediated first phase and the immune-mediated second phase. DeltaHCV RNA less than 50% (group 1); DeltaHCV RNA greater than 50% but less than 90% (group 2); and DeltaHCV RNA > or =90% (group 3) were differentiated. The PePHD region was highly conserved; the few mutations (5 patients) did not correlate with DeltaHCV RNA or sustained virologic response to IFN-alpha. Within the flanking regions before and after PePHD (codons 618-681) 72 of 81 patients (89%) had 2.6+/-0.17 mutations (median, 3; range, 1-8) that did not correlate with treatment response. Sequence analysis of the NS5A protein (codons 2,209-2,274, including interferon sensitivity determining region [ISDR]) in 39 of 81 patients showed a higher mean number of mutations in the ISDR (codons 2,209-2,248) in groups 2 (1.28+/- 0.43 [n = 18]) and 3 (1.89+/-0.54 [n = 9]) than in group 1 (0.67+/- 0.19 [n = 12]; P =.049 group 1 vs. 3) and a mutant type ISDR (e.g., > or =4 mutations) was significantly more frequent in sustained virologic responders than in nonresponders or relapsers (2 of 4 [50%] vs. 2 of 35 [6%]; P =.045). Thus, NS5A appears to be functionally relevant in IFN-alpha-induced effector reactions.

Topics: Amino Acid Sequence; Antiviral Agents; eIF-2 Kinase; Female; Gene Frequency; Hepacivirus; Hepatitis C; Humans; Interferon-alpha; Male; Middle Aged; Mutation; Peptide Fragments; Phylogeny; Protein Structure, Secondary; Time Factors; Viral Envelope Proteins; Viral Nonstructural Proteins; Viremia

The NS5A and the E2 proteins of hepatitis C virus (HCV)-1b can bind and inhibit in vitro the interferon (IFN)-induced cellular kinase PKR. The role of such interaction in modulating the antiviral effect of IFN is still controversial. We have analyzed the E2 and the NS5A sequences in HCV-1b-infected patients treated with IFN to assess whether and how different combinations of wild-type and mutant proteins correlated with early and long-term virological response.. In 30 patients, sequences of pretreatment and on-treatment E2-PePHD and NS5A-PKR binding domain (including the putative ISDR) were analyzed in parallel by sequencing cDNA-polymerase chain reaction products and up to 25 independent clones.. The E2-PePHD sequence was highly conserved with a homogeneous quasispecies and was identical in 29 of 30 cases with no association with the pattern of response and no evidence of evolution during therapy. Patients with a mutated NS5A-ISDR had a higher rate of early virological response (67%) than cases with wild-type ISDR (17%). This association was lost in long-term responders (33% vs. 17%).. Although the highly conserved E2-PePHD motif might contribute to reduce IFN responsiveness, variations within this region do not seem to play a role in modulating IFN sensitivity. The NS5A-ISDR sequence influenced the early, but not the sustained response, to IFN, suggesting that other factors may be more important for the long-term outcome of therapy.

Topics: Adult; Amino Acid Sequence; Antiviral Agents; Conserved Sequence; Drug Resistance; eIF-2 Kinase; Female; Hepatitis C; Humans; Interferons; Male; Middle Aged; Molecular Sequence Data; Mutation; Time Factors; Viral Envelope Proteins; Viral Nonstructural Proteins

In order to study the significance of HCVE2/NS1 relative conservation region antigen.. EIA was established with HCVE2/NS1 relative conservation polypeptide antigen, as well as serum HCV RNA and ALT in patients with HCV infection.. The positive rate of 96 cases patients who were infected by HCV is 41.67%, acute 13.55% and chronic 25.04%, and we found that there were 3 cases weak positive in 40 healths; Agreement rate is 62.5% in 40 cases of the positive of anti-E2/NS1 in sera with HCVRNA, and they have relation. In ALT level in sera, acute infection of HCV is higher than chronic (P < 0.05).. There are virus necleic acid in sera when anti-HCVE2/NS1 appearence positive; however, antibody of anti-E2/NS1 relative conservation region has the some neutralive actions, and this humor immunity may have the actions in clearing virus, but wether anti-body of anti-E2/NS1 relative conservation of HCV is or not neutralive antibody well be investigated further.

Topics: Adult; Alanine Transaminase; Enzyme-Linked Immunosorbent Assay; Female; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Male; Viral Envelope Proteins

Evidence from clinical and experimental studies of human and chimpanzees suggests that hepatitis C virus (HCV) envelope glycoprotein E2 is a key antigen for developing a vaccine against HCV infection. To identify B-cell epitopes in HCV E2, six murine monoclonal antibodies (MAbs), CET-1 to -6, specific for HCV E2 protein were generated by using recombinant proteins containing E2t (a C-terminally truncated domain of HCV E2 [amino acids 386 to 693] fused to human growth hormone and glycoprotein D). We tested whether HCV-infected sera were able to inhibit the binding of CET MAbs to the former fusion protein. Inhibitory activity was observed in most sera tested, which indicated that CET-1 to -6 were similar to anti-E2 antibodies in human sera with respect to the epitope specificity. The spacial relationship of epitopes on E2 recognized by CET MAbs was determined by surface plasmon resonance analysis and competitive enzyme-linked immunosorbent assay. The data indicated that three overlapping epitopes were recognized by CET-1 to -6. For mapping the epitopes recognized by CET MAbs, we analyzed the reactivities of CET MAbs to six truncated forms and two chimeric forms of recombinant E2 proteins. The data suggest that the epitopes recognized by CET-1 to -6 are located in a small domain of E2 spanning amino acid residues 528 to 546.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; CHO Cells; Cricetinae; Enzyme-Linked Immunosorbent Assay; Epitopes, B-Lymphocyte; Female; Hepacivirus; Hepatitis C; Humans; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Viral Envelope Proteins

The relationship of viral persistence, the immune response to hepatitis C virus (HCV) envelope proteins, and envelope sequence variability was examined in chimpanzees. Antibody reactivity to the HCV envelope proteins E1 or E2 was detected by enzyme-linked immunosorbent assay (ELISA) in more than 90% of a human serum panel. Although the ELISAs appeared to be sensitive indicators of HCV infection in human serum panels, the results of a cross-sectional study revealed that a low percentage of HCV-inoculated chimpanzees had detectable antibody to E1 (22%) and E2 (15%). Viral clearance, which was recognized in 28 (61%) of the chimpanzees, was not associated with an antibody response to E1 or E2. On the contrary, antibody to E2 was observed only in viremic chimpanzees. A longitudinal study of animals that cleared the viral infection or became chronically infected confirmed the low level of antibody to E1, E2, and the HVR-1. In 10 chronically infected animals, the sequence variation in the E2 hypervariable region (HVR-1) was minimal and did not coincide with antibody to E2 or to the HVR-1. In addition, low nucleotide and amino acid sequence variation was observed in the E1 and E2 regions from two chronically infected chimpanzees. These results suggest that mechanisms in addition to the emergence of HVR-1 antibody escape variants are involved in maintaining viral persistence. The significance of antibodies to E1 and E2 in the chimpanzee animal model is discussed.

Topics: Amino Acid Sequence; Animals; Cell Line; Cross-Sectional Studies; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Longitudinal Studies; Molecular Sequence Data; Pan troglodytes; Recombinant Fusion Proteins; Sequence Analysis, DNA; Viral Envelope Proteins; Viremia; Virus Latency

We characterized immunoreactive B-cell domains of hepatitis C virus (HCV) envelope proteins E1 and E2 by a peptide ELISA using sera of patients who were infected by the same isolate of HCV (HCV-AD78).. Fifty-four overlapping peptides which corresponded to the sequence of E1 and E2 of isolate HCV-AD78 were used to detect specific antibodies. Three groups of HCV-AD78 related sera were analyzed. Two groups were from sera obtained at early time points of infection (months 4-15) from patients who later resolved infection (group A), or who later developed chronic disease (group B). Group C sera were from later time points of chronic disease. As a control, sera of chronic HCV patients who did not have HCV-AD78 infection were also analyzed (group D).. In group A, 25 of the 54 peptides produced OD405 above the cut-off, whereas 17 peptides produced such values in group B. Only 10 and 3 peptides yielded such values in groups C and D, respectively. The overall prevalence of antibodies against peptides was high in the early phase of infection (means of 28.7+/-14.8% and 25.9+/-14.5% in groups A and B, respectively). At later time points of chronic infection (group C), the overall prevalence was lower (mean 18.6+/-15.4%). Group D sera produced the lowest overall prevalence (mean 13.2+/-14.1%). Three peptides, covering aa271-290, aa481-500 and aa551-570, were recognized significantly more frequently (p<0.05) by group A sera than group B sera.. We conclude that more linear epitopes of the HCV envelope are recognized with a high prevalence of antibodies, as was suggested previously. However, most B-cell domains of the HCV envelope induce a similarly high antibody response in patients who resolve infection or develop chronic disease.

Topics: Adult; Amino Acid Sequence; B-Lymphocytes; Female; Hepatitis C; Hepatitis C Antibodies; Humans; Immunodominant Epitopes; Molecular Sequence Data; Peptide Fragments; Time Factors; Viral Envelope Proteins

We have studied the evolution of hepatitis C virus (HCV) from a common source following serial transmission from contaminated batches of anti-D immunoglobulin. Six secondary recipients were each infected with virus from identifiable primary recipients of HCV-contaminated anti-D immunoglobulin. Phylogenetic analysis of virus E1/E2 gene sequences [including the hypervariable region (HVR)] and part of NS5B confirmed their common origin, but failed to reproduce the known epidemiological relationships between pairs of viruses, probably because of the frequent occurrence of convergent substitutions at both synonymous and nonsynonymous sites. There was no evidence that the rate at which the HCV genome evolves is affected by transmission events. Three different mechanisms appear to have been involved in generating variation of the hypervariable region; nucleotide substitution, insertion/deletion of nucleotide triplets at the E1/E2 boundary and insertion of a duplicated segment replacing almost the entire HVR. These observations have important implications for the phylogenetic analysis of HCV sequences from epidemiologically linked isolates.

Topics: Adolescent; Adult; Aged; Amino Acid Sequence; Base Sequence; Blood Transfusion; Child; Drug Contamination; Evolution, Molecular; Female; Genetic Variation; Genome, Viral; Hepacivirus; Hepatitis C; Humans; Infectious Disease Transmission, Vertical; Ireland; Male; Molecular Sequence Data; Mutation; Phylogeny; Retrospective Studies; Rho(D) Immune Globulin; Viral Envelope Proteins

A systematic virological follow-up of hemodialysis patients identified 11 cases of de novo hepatitis C virus (HCV) infection in the same unit that were not due to blood transfusion. There were three groups of infection, each occurring within a period of 3 months: four infections with genotype 1b, two infections with genotype 1b, and five infections, four with genotype 1a and one with genotype 5a. The possibility of patient-to-patient transmission was addressed by sequencing the first hypervariable region of the HCV genome in sera taken shortly after infection. Phylogenetic analysis indicated clustering of most of the cases of de novo infections. Sequence homologies identified potential contaminators among already infected patients. All patients who were infected with closely related HCV isolates were found to have been treated in the same area and during the same shift or on the previous one. These infections could have been due to occasional breaches of the usual hygiene measures. Strict adhesion to hygiene standards and routines, continuously supervised, remains the key rule in the management of dialysis patients. Nevertheless, the isolation of patients with HCV could reduce the risk of infection because occasional lapses of preventive hygiene measures or unpredictable accidents can always take place in a hemodialysis unit. This policy needs to be evaluated by large-scale prospective studies.

Topics: Adult; Aged; Cross Infection; Female; France; Genetic Variation; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hospitals, University; Humans; Male; Middle Aged; Molecular Sequence Data; Phylogeny; Polymerase Chain Reaction; Prospective Studies; Renal Dialysis; RNA, Viral; Viral Envelope Proteins

A chimpanzee was immunized with two recombinant envelope glycoproteins E1 and E2 of hepatitis C virus (HCV), strain HCV-N2, and the hypervariable region 1 (HVR1) peptides of a different isolate, HCV-#6, then received an intravenous inoculation of 10 chimpanzee infectious doses of HCV-#6. With high humoral immune response against E1 and E2 but a low response against HVR1, the vaccinee became infected with the HCV. However, after increasing the titer of anti-HVR1 against HCV-#6, the vaccinee showed protection. Neutralization of HCV-#6 with the antiserum from this protected vaccinee was achieved by inoculation of this mixture into another chimpanzee. These results suggest that vaccination with a peptide-vaccine of homologous HVR1 is effective in the chimpanzee.

Topics: Amino Acid Sequence; Animals; Antibody Formation; Genetic Variation; Hepacivirus; Hepatitis C; Molecular Sequence Data; Pan troglodytes; Sequence Alignment; Sequence Homology, Amino Acid; Time Factors; Vaccines, Synthetic; Viral Envelope Proteins; Viral Hepatitis Vaccines

Hepatitis C virus (HCV) load is one of the most important predictive factors of response to interferon treatment. However, little is known about the mode and determinants of viremia. The mode of viremia was investigated in 78 patients with chronic HCV genotype 1b infection during 1-2 years follow up. Virus load, determined by a branched chain DNA amplification assay, was stable in 73 of 78 (93.6%) patients, whereas 5 (6.4%) showed marked fluctuation (from undetectable level to more than 10 Meq/ml) in viral titer. To study the mechanisms mediating fluctuations in viral titer, amino acid sequences of two regions were examined; hypervariable region (HVR) 1 and the interferon sensitivity determining region (ISDR). Multiple amino acid substitutions were observed in HVR 1 but no relationship was evident between substitutions and virus titers. In contrast, no amino acid substitutions were observed in the ISDR in any patients with stable virus titer during a follow-up period of 12-24 months (7-24 samples) or in one patient who was observed for 15 years. Interestingly, multiple amino acid substitutions in the ISDR appeared in only two of the five patients with marked titer fluctuation, when the virus decreased markedly. Alanine aminotransferase levels in these five patients correlated with viral load. The data suggest that amino acid substitutions in HVR1 and ISDR are not essential for changes in viral titer. Possible mechanisms of fluctuations of viral titer and amino acid substitutions in the ISDR accompanying marked reductions in viral load are discussed.

Topics: Alanine Transaminase; Amino Acid Sequence; Amino Acid Substitution; Antiviral Agents; DNA, Viral; Follow-Up Studies; Genetic Variation; Hepacivirus; Hepatitis C; Humans; Interferons; Molecular Sequence Data; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Time Factors; Viral Envelope Proteins; Viral Load; Viral Nonstructural Proteins

E2 glycoprotein of hepatitis C virus was expressed in mammalian cell and purified for detection of antibody against E2 in hepatitis C patients.. E2/NS1 gene derived from HCV was inserted into expression vector containing six His tag. The recombinant plasmid was transfected into mammalian cells to express E2 glycoprotein expression. E2 glycoprotein was purified by affinity chromophotography. The purified protein was used to establish EIA method for detection of antibodies against E2 in hepatitis C patients.. Expressed E2 glycoprotein was 7.0 x 10(4). Purification of the purified E2 protein was 90.2%. Twenty-nine patients were anti-E2 antibody positive(82.9%).. It was the first time to establish EIA method for detection of anti-E2 antibody by purified E2 glycoprotein in China. E2 glycoprotein expressed in mammalian cells had good immunogenity and could increase the sensibility of anti-HCV detection. It suggests that E2 glycoprotein may be useful for development of new anti-HCV reagents.

Topics: Hepatitis C; Hepatitis C Antibodies; Humans; Immunoenzyme Techniques; Recombinant Proteins; Serologic Tests; Viral Envelope Proteins

To explore the possibility and the efficacy of immune responses in mice inoculated with recombinant plasmid pCD-HCV1 and to lay a foundation for HCV nucleic acid vaccine development in the future.. The gene fragment coding C and E regions of HCV-II (type I b) was inserted into pCD-SR alpha 1 expression vector and formed pCD-HCV1 and then was injected into quadriceps muscles of Balb/c mouse. Serum anti-HCV level of mice was tested by ELISA (A value). Spleen cells proliferation responses to HCV antigens were detected by 3H-TdR incorporation (cpm).. Balb/c mice immunized with recombinant plasmid pCD-HCV1 three or four times can generate specific antibody responses to HCV antigens and the antibody levels gradually ascend to the plateaus and did not have the trend of descending in 18 weeks detected. The serum antibodies in mice immunized by recombinant plasmid pCD-HCV1 were 100 percent positive when the serum were diluted 40 times and the positive rate of antibody still were 16.6 percent positive when the serum were diluted 320 times. Balb/c mice immunized with recombinant plasmid pCD-HCV1 (100 micrograms, 50 micrograms 10 micrograms/mouse three times respectively) can elicit antibody responses to HCV antigens and the antibody levels of three groups were 0.70 +/- 0.07, 0.33 +/- 0.04 and 0.11 +/- 0.09 respectively. Spleen cells of Blab/c mice injected with pCD-HCV1 three times were induced to produce proliferation responses to HCVc + e specific antigens.. These results demonstrated that constructs expressioning HCV core and envelope proteins can generate anti-HCVc + e specific antibody responses and lymphoproliferation responses in mice, which suggested it to be possible to elicit immune responses to viral epitopes from HCV via DNA immunization with HCV-DNA recombinant and to warrant further investigation as a potential vaccine against HCV infections.

Topics: Animals; DNA, Recombinant; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Male; Mice; Mice, Inbred BALB C; Plasmids; Vaccination; Viral Core Proteins; Viral Envelope Proteins

Direct DNA intramuscular or intradermal injection of plasmids containing viral genes under the control of viral promoters is an efficient means of stimulating both class I and class II-mediated antiviral responses. Viral hepatitis B and C are suitable candidates for this approach, particularly as therapeutic immunogens for chronically infected individuals. Several groups have shown that the S gene of HBV is expressed in murine muscle and stimulates a high titre and long-lasting anti-HBs response. Uniquely, CD8+ CTL responses are also induced to HBsAg. No vaccine exists for HCV. Therefore the structural genes (C + E1 + E2) have been cloned as a 2,831 bp fragment from a genotype la isolate into the vector pcDNA3. The resulting plasmid DNA was injected directly into the quadriceps muscle of three-week-old BALB/c mice. Intracellular-expressed E1 and E2 proteins thus represent the complete spectrum of native structural epitopes, including those dependent on glycosylation and protein folding. Mouse antisera were tested for reactivity against conserved sequences using overlapping 7-mer peptides. Two conserved, overlapping epitopes were identified in E2 spanning residues 581-591 and 590-603. This domain represents one of seven major E2 antigenic domains recognized by HCV human antibodies, one of three with antigenic homologies to related flavivirus proteins. Thus antigen is presented with high efficiency following DNA injection and offers the potential of high rates of seroconversion and virus clearance in those predisposed to virus-induced chronic liver disease.

Topics: Animals; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay; Genes, Viral; Hepacivirus; Hepatitis C; Humans; Male; Mice; Mice, Inbred BALB C; Promoter Regions, Genetic; Vaccines, DNA; Viral Envelope Proteins; Viral Hepatitis Vaccines; Viral Structural Proteins

To study hepatitis C virus (HCV) genetic mutation during interferon (IFN) therapy, the temporal changes in HCV quasispecies heterogeneity were compared before and after treatment for nine patients infected with HCV genotype 1, including four nonresponders, four responders who relapsed after therapy, and one responder who experienced a breakthrough of viremia during therapy. Nine untreated patients with an average time between specimens of 8.4 years served as controls. Sequences from the second envelope glycoprotein gene hypervariable region 1 (HVR1) and the putative IFN sensitivity-determining region (ISDR) of the nonstructural NS5A gene were analyzed by heteroduplex mobility assays and nucleotide sequencing. A strong positive correlation was found between the percent change in a heteroduplex mobility ratio (HMR) and percent change in nucleotide sequence (r = 0.941, P < 0.001). The rate of fixation of mutations in the HVR1 was significantly higher for IFN-treated patients than for controls (6.97 versus 1.31% change in HMR/year; P = 0.02). Similarly, a higher rate of fixation of mutations was observed in the ISDR for IFN-treated patients than for untreated controls, although the result was not significant (1.45 versus 0.15 amino acid changes/year; P = 0.12). On an individual patient basis, IFN therapy was associated with measurable HVR1 and ISDR mutation in nine of nine (100%) and two of nine (22.2%) patients, respectively. Evolution to IFN-resistant ISDR sequences was observed in only one of nine IFN-treated patients. These data suggest that IFN therapy frequently exerts pressure on the HCV envelope region, while pressure on the ISDR was evident in only a subset of patients. Thus, the selection pressures evoked on HCV genotype 1 quasispecies during IFN therapy appear to differ among different patients.

Topics: Amino Acid Sequence; Antiviral Agents; Evolution, Molecular; Genetic Variation; Hepacivirus; Hepatitis C; Humans; Interferons; Molecular Sequence Data; Mutation; Nucleic Acid Heteroduplexes; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Time Factors; Viral Envelope Proteins; Viral Nonstructural Proteins

The genomic sequence of an Australian isolate of hepatitis C virus (HCV) was determined from overlapping cDNA clones obtained from a small amount (1.2 ml) of serum from a single individual with hepatitis C. The isolate (HCV-A) comprises 9379 nucleotides (nt) including 324 nt of a 5' untranslated region (5'UTR), a single long open reading frame of 9033 nt encoding a polyprotein of 3010 amino acids (aa), and 22 nt of a 3' untranslated region (3'UTR). Sequence analysis of a 251 nt region within the 5'UTR and a 222 nt region within NS5B showed the genotype of HCV-A to be subtype 1b. A striking difference in the amino acid sequence of the hypervariable region 1 (HVR-1), and not in the surrounding sequence, was seen in cDNA clones synthesised from serum taken 52 weeks after the initial sample, indicating a significant population diversity of HCV genomes.

Topics: Amino Acid Sequence; Australia; Base Sequence; Cloning, Molecular; DNA, Viral; Genome, Viral; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Viral Envelope Proteins

The long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed.

Topics: Amino Acid Sequence; Cohort Studies; Evolution, Molecular; Female; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Phylogeny; Sequence Analysis; Viral Envelope Proteins

GB virus C/hepatitis G virus (GBV-C/HGV) is related distantly to hepatitis C virus (HCV). HCV has a hypervariable region (HVR), and exists as quasispecies in vivo. Although GBV-C/HGV also has replaceable amino acids in the presumed antigenic region, the existence and fluctuation of population of heterogeneous virus have not been evaluated. In this study, the heterogeneity of GBV-C/HGV and HCV was investigated by the single-strand conformation polymorphism (SSCP) analysis in six concomitantly infected patients. Two patients were observed for 4 years without any treatment, and four were treated with interferon-alpha (IFN). By SSCP analysis, amplicons of GBV-C/HGV RNA were separated into 1-5 bands on gels for each patient. The amplicons had different nucleotide but the same amino acid sequences in the presumed antigenic region. The amplicons of HCV RNA, separated into 1-4 bands, had different nucleotide and amino acid sequences in the HVR. In the two patients without treatment, the predominant strain of GBV-C/HGV was unchanged for the 4 years. In the four patients administered IFN, some strains of GBV-C/HGV disappeared after IFN therapy, whereas other strains persisted. The mean genetic distance among GBV-C/HGV strains represented by SSCP analysis was significantly lower than that of HCV (P < 0.05). The data indicate that: 1) GBV-C/HGV can be devoid of antigenic drift unlike HCV; 2) GBV-C/HGV has no HVR as seen in HCV in the presumed antigenic region; and 3) the sensitivity to IFN differs among GBV-C/HGV strains in the same hosts, as with HCV.

Topics: Adult; Aged; Amino Acid Sequence; Antiviral Agents; Base Sequence; DNA, Viral; Female; Flaviviridae; Genetic Heterogeneity; Hepacivirus; Hepatitis C; Hepatitis, Viral, Human; Humans; Interferon-alpha; Male; Middle Aged; Molecular Sequence Data; Polymorphism, Single-Stranded Conformational; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Viral Envelope Proteins

Hepatitis C virus (HCV) infection is a dynamic process during which molecular variants are continuously selected as the result of virus adaptation to the host. Understanding the nature of HCV genetic variation is central to current theories of pathogenesis and immune response. We prospectively studied hypervariable region 1 (HVR1) variation in the E2 gene of 36 hepatitis C patients, including 10 asymptomatic carriers, followed up for 1 to 2 years. Sequence changes in single and consecutive serum samples were assessed and correlated with clinical and virological parameters of liver disease. A region of the E1 gene was sequenced for comparison in 3 subjects. HVR1 heterogeneity at single time points widely varied in individual patients, did not increase cumulatively over the follow-up period, and did not correlate with HVR1 evolutionary rates. Conversely, the process of HVR1 sequence diversification, although differed considerably among patients, was stable over time and directly correlated with infections by HCV type 2, lower alanine aminotransferase (ALT) levels, and absence of cirrhosis. HCV carriers showed the highest HVR1 variation rates. Our findings indicate that HVR1 variation has an adaptive significance and is associated with favorable features of liver disease and suggest that prospective, rather than static, observations are required to model the process of HCV variation.

Topics: Adult; Aged; Cloning, Molecular; Female; Genes, Viral; Genetic Variation; Genome, Viral; Hepacivirus; Hepatitis C; Humans; Male; Middle Aged; Viral Envelope Proteins

To clarify the nature of serum anti-hypervariable region 1 (HVR1) antibodies in patients infected with hepatitis C virus (HCV), we assessed the reactivity of 21 patients' sera with 42 HVR1 proteins by Western blot. HVR1 was expressed as fusion proteins with glutathione S-transferase (GST). The patients' sera reacted with variable percentages of the HVR1 proteins, and always reacted with HVR1 proteins of the different genotype. In the genotype-1b-infected patients, the percentage of genotype-1b HVR1 proteins reactive with serum correlated significantly with viral loads; the sera reactive with the higher percentages of HVR1 proteins contained the larger viral loads. In addition, it was significantly lower in the responders of interferon (IFN) therapy than in nonresponders. The competition assays indicated that multiple fractions of anti-HVR1 antibodies with different specificity in a serum reacted with different HVR1 proteins, and that, additionally, a single fraction of antibodies often reacted with more than one HVR1 protein through a similar amino acid sequence. In conclusion, serum anti-HVR1 antibodies were broadly reactive by the mechanism of both the cross-reactivity of a single fraction of anti-HVR1 antibodies with more than one HVR1 protein and the presence of multiple fractions of anti-HVR1 antibodies with different specificity in a serum. In genotype-1b-infected patients, the broad reactivity of serum anti-HVR1 antibodies correlated with viral loads and response to IFN. Further studies are necessary to elucidate the correlation among the broad reactivity of sera with multiple HVR1 proteins and clinical features of chronic hepatitis C patients.

Topics: Adult; Antibodies, Viral; Antibody Specificity; Antiviral Agents; Female; Genome, Viral; Hepacivirus; Hepatitis C; Humans; Interferons; Male; Middle Aged; Molecular Sequence Data; Viral Envelope Proteins; Viral Load

The hypervariable region 1 (HVR-1) of the putative envelope encoding E2 region of hepatitis C virus (HCV) RNA was analyzed in sequential samples from three patients with acute type C hepatitis infected from different sources to address (i) the dynamics of intrahost HCV variability during the primary infection and (ii) the role of host selective pressure in driving viral genetic evolution. HVR-1 sequences from 20 clones per each point in time were analyzed after amplification, cloning, and purification of plasmid DNA from single colonies of transformed cells. The intrasample evolutionary analysis (nonsynonymous mutations per nonsynonymous site [Ka], synonymous mutations per synonymous site [Ks], Ka/Ks ratio, and genetic distances [gd]) documented low gd in early samples (ranging from 2. 11 to 7.79%) and a further decrease after seroconversion (from 0 to 4.80%), suggesting that primary HCV infection is an oligoclonal event, and found different levels and dynamics of host pressure in the three cases. The intersample analysis (pairwise comparisons of intrapatient sequences; rKa, rKs, rKa/rKs ratio, and gd) confirmed the individual features of HCV genetic evolution in the three subjects and pointed to the relative contribution of either neutral evolution or selective forces in driving viral variability, documenting that adaptation of HCV for persistence in vivo follows different routes, probably representing the molecular counterpart of the viral fitness for individual environments.

Topics: Adolescent; Adult; Amino Acid Sequence; Biological Evolution; Female; Hepacivirus; Hepatitis C; Humans; Male; Molecular Sequence Data; Viral Envelope Proteins

We previously found two cell lines (MT-2 and PH5CH) that were susceptible to hepatitis C virus (HCV) infection. Analysis of the infectivity of sera from HCV-positive blood donors for MT-2 and PH5CH cells suggested the cell tropism of HCV. To investigate further the cell tropism of HCV, the dynamics of HCV populations during culture were examined using three MT-2 clones and three PH5CH clones, infected with inoculum 1B-2. To type HCV populations in these infected cells, the HCV hypervariable region 1 (HVR1) in these cloned cells was characterized by sequence analysis and HpaII digestion analysis, which could distinguish three major HVR1 types (I, II and III) derived from the inoculum 1B-2. It was found that genomes containing HVR1 type I became predominant in MT-2 clones, and genomes containing HVR1 type II became predominant in PH5CH clones during culture after inoculation. These results suggest that inoculum 1B-2 contains both lymphotropic and hepatotropic HCV species, which can be distinguished by HVR1 type. To search for cell type-specific sequences in regions other than HVR1, three HCV cDNA clones (3.4 kb of the 5' noncoding region to the nonstructural 2 region) containing HVR1 type I obtained from HCV-infected MT-2C cells, and three HCV cDNA clones containing HVR1 type II obtained from HCV-infected PH5CH7 cells were sequenced. Following a comparison of the sequences, 11 amino acids were identified as candidates for determinants of the cell tropism of HCV.

Topics: Amino Acid Sequence; Cell Line, Transformed; Deoxyribonuclease HpaII; DNA Primers; Hepacivirus; Hepatitis C; Humans; Liver; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Viral; T-Lymphocytes; Viral Envelope Proteins; Viral Nonstructural Proteins

Chronic hepatitis C virus (HCV) infection occurs in about 3 percent of the world's population and is a major cause of liver disease. HCV infection is also associated with cryoglobulinemia, a B lymphocyte proliferative disorder. Virus tropism is controversial, and the mechanisms of cell entry remain unknown. The HCV envelope protein E2 binds human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Binding of E2 was mapped to the major extracellular loop of CD81. Recombinant molecules containing this loop bound HCV and antibodies that neutralize HCV infection in vivo inhibited virus binding to CD81 in vitro.

Topics: Amino Acid Sequence; Animals; Antibodies; Antibodies, Viral; Antigens, CD; Cell Line; DNA, Complementary; Gene Library; Hepacivirus; Hepatitis C; Humans; Liver; Lymphocytes; Membrane Proteins; Mice; Molecular Sequence Data; Pan troglodytes; Recombinant Fusion Proteins; Recombinant Proteins; Sequence Alignment; Tetraspanin 28; Tumor Cells, Cultured; Viral Envelope Proteins

The absence of readily available animal and cell culture models for hepatitis C virus (HCV) replication has bottlenecked research on protective immunity to HCV infection. Antibodies reactive with HCV virions in vitro are assumed to be candidates for neutralizing or inhibitory antibodies against HCV. To find potentially neutralizing or inhibitory antibody candidates, anti-C, anti-E1, anti-E2, and anti-HVR1 antisera acquired from mice immunized with corresponding recombinant proteins or synthetic peptides were used to capture HCV viral particles in vitro based on antibody-virus interaction assays. Both anti-E2 and anti-HVR1 antibodies effectively captured HCV in vitro. Furthermore, it was found that anti-E2 and anti-HVR1 antibodies could immunoprecipitate an isolate of HCV unrelated to the original antigenic HCV isolate. ELISA confirmed that anti-HVR1 antibodies cross-reactively bind to these unrelated HVR1 peptides. These findings suggest that anti-E2 and anti-HVR1 antibodies induced in mice have the ability to bind with HCV particles in an isolate cross-reactive manner and highlight the possible application of combining several sequences of HVR1 to generate broadly reactive anti-HVR1 antibodies.

Topics: Amino Acid Sequence; Animals; Antigen-Antibody Complex; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Mice; Mice, Inbred Strains; Molecular Sequence Data; Phylogeny; Precipitin Tests; RNA, Viral; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Viral Envelope Proteins; Viral Proteins

Hepatitis C virus (HCV) RNA extracted from serum of a patient infected with HCV in Beijing was converted to cDNA by reverse transcription. HCV E2/NS1 gene (approximately 930 bp) was amplified by nested polymerase chain reaction and was inserted into pGEM-T plasmid vector. The 431 bp nucleotide sequence at 5' terminus of this gene was determined by dideoxy-mediated chain termination method. The result of comparison of this sequence and sequences of another 9 HCV isolates suggested that the isolate we obtained was highly homologous to HCV genotype II. There are two hypervariable regions (HVR) at the N terminus of envelope protein encoded by HCV E2/NS1 gene. Several relatively conserved amino acid residues and areas located in and out of HVR were also observed. They are concerned in maintenance of spatial conformation of HCV envelope protein, but their immune function remains to be further studied.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA, Complementary; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Sequence Alignment; Viral Envelope Proteins; Viral Nonstructural Proteins

The antibody response to the hypervariable region of the E2 protein (HVR1) of hepatitis C virus (HCV) was studied in 5 patients who were infected by a common virus strain during an outbreak in a hemodialysis unit. Two patients resolved the infection, while 3 developed chronic HCV infection. For studying the antibody response to HVR1 during the early phase of infection, a Western blot assay using recombinant phage displaying HVR1 was developed. The 2 patients with resolving infection had a more rapid antibody response to HVR1 than did the patients developing chronic infection. Anti-HVR1 antibodies were repeatedly absent in 1 of the chronically infected patients. Antibodies to recombinant E2 protein occurred later than the anti-HVR1 antibodies and did not correlate with resolution of the infection. Thus, the present results suggest that early appearance of antibodies to the HVR1 may predict clearance of HCV infection.

Topics: Amino Acid Sequence; Antibodies, Viral; Bacteriophages; Blotting, Western; Chronic Disease; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay; Hepacivirus; Hepatitis C; Humans; Immunoglobulin G; Molecular Sequence Data; Recombinant Proteins; Renal Dialysis; Viral Envelope Proteins

Hepatitis C virus (HCV), a major causative agent of non-A, non-B chronic hepatitis, is also suggested to be associated with extrahepatic manifestations such as mixed cryoglobulinemia and glomerulonephritis. Two independent lines of transgenic mice carrying the HCV envelope genes have been shown previously to express the HCV envelope proteins in organs, including the liver and salivary glands, which results in no pathological changes in the liver. Further analysis of these animals now has revealed that they develop an exocrinopathy involving the salivary and lachrymal glands. This pathology resembles Sjogren syndrome, which also is suggested to have a possible association with chronic hepatitis C. These observations suggest that HCV might be involved in the pathogenesis of sialadenitis in humans and that this transgenic mouse system would be a good animal model for the study of HCV infection.

Topics: Age Factors; Animals; Disease Models, Animal; Female; Genes, Viral; Hepacivirus; Hepatitis C; Lacrimal Apparatus; Liver; Male; Mice; Mice, Transgenic; Salivary Glands; Sialadenitis; Sjogren's Syndrome; Viral Envelope Proteins

Hepatitis C virus (HCV) results frequently in chronic hepatitis and its sequelae liver cirrhosis and hepatocellular carcinoma. Interferon-alpha is at present the most effective treatment, resulting in a sustained response in about 20-25% of patients. HCV genotype, titer and quasispecies determine the success of treatment. In this study, fluorescent single strand conformation polymorphism (f-SSCP) was evaluated for the analysis of HCV quasispecies. Two sera from a chronically HCV-infected patient, obtained 6 years apart, were examined. The hypervariable region I (HVRI) of the HCV genome was amplified by reverse transcription and PCR. The PCR products were cloned and sequenced or fluorescein-labeled and subjected to f-SSCP. Both methods demonstrated a single HCV species in the early serum and multiple quasispecies in the late serum. Single clones of the heterogeneous virus population were used to optimize conditions for f-SSCP. The most important factors were the gel temperature and virus titer. At the optimal running temperature one base exchange in 218 bases was detectable. Repeat extractions and amplifications gave identical results. Dilution of the serum containing multiple quasispecies resulted in a 'loss' of species. Provided the running temperature is optimal and virus titer is sufficient, f-SSCP is shown to be fast and reliable for HCV quasispecies analysis.

Topics: Amino Acid Sequence; Base Sequence; DNA, Viral; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; RNA, Viral; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Species Specificity; Viral Envelope Proteins

Antibody response to the E2/NS1 protein of the hepatitis C virus (HCV) was studied in 26 patients with post-transfusion acute hepatitis C. Second-generation HCV (HCV-2) antibody, E2/NS1 antibody and HCV-RNA were measured in serial serum samples taken within 1 month and 3, 6 and 12 months after the onset of acute hepatitis C. The HCV genotype was also tested to study its clinical significance. Of 26 patients, eight showed normalization of alanine aminotransferase (ALT) and clearance of HCV-RNA (resolved group). In the remaining 18 patients, HCV viraemia and ALT abnormality (except one patient) continued for more than 3 years (unresolved group). Both HCV-2 and E2/NS1 antibodies were positive at least once in all patients. The prevalence of E2/NS1 antibody was significantly (P < 0.05) higher in the resolved group (88%) than in the unresolved group (39%) in the period within 1 month of onset; the prevalence was similar between the two groups thereafter. The prevalence of HCV-2 antibody did not differ between the two groups at any point. The HCV genotype was not related to the chronicity of acute hepatitis C. In conclusion, the E2/NS1 antibody appeared in all patients with acute hepatitis C and was associated with the clearance of HCV.

Topics: Acute Disease; Adult; Alanine Transaminase; Blood Transfusion; Case-Control Studies; Clinical Enzyme Tests; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Prevalence; RNA, Viral; Seroepidemiologic Studies; Time Factors; Viral Envelope Proteins; Viral Nonstructural Proteins

The viral variability of 5 hepatitis C virus (HCV)-infected immunocompromised patients was analyzed and compared with that in isolates from immunocompetent subjects. The patients were followed longitudinally with regard to changes in hypervariable region 1 (HVR1) of HCV using a direct DNA sequencing approach. For the immunocompromised patients, viral nucleotide sequence variability was markedly lower than in immunocompetent HCV-positive patients. For 1 agammaglobulinemic patient and 1 AIDS patient, no variation in the major amino acid sequence of HCV HVR1 could be observed, while another agammaglobulinemic patient exhibited transient variations and amino acid substitutions despite the lack of functioning humoral immune response. The study supports the general hypothesis of humoral immune selection as the main force of sequence variation in the HVR1 region but suggests that other selection mechanisms may contribute to modulation of the composition of the viral population.

Topics: Amino Acid Sequence; Base Sequence; Chronic Disease; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunocompromised Host; Molecular Sequence Data; Peptide Fragments; Viral Envelope Proteins

Epitopes of hypervariable region 1 (HVR1) were mapped by enzyme-linked immunosorbent assay using follow-up sera of patients, all of whom were infected with the same isolate of hepatitis C virus (HCV). Our results suggest that (i) an early appearance (up to month 13 postinfection) of antibodies directed to the N terminus of HVR1 is associated with acute self-limiting infections of HCV and (ii) isolate-independent antibodies which are mainly directed to the C terminus of HVR1 seem to persist in chronically infected patients. The relevance of HVR1-specific antibodies for neutralization was evaluated by characterization of a rabbit serum.

Topics: Acute Disease; Amino Acid Sequence; Animals; Chronic Disease; Epitope Mapping; Hepatitis C; Hepatitis C Antibodies; Humans; Molecular Sequence Data; Peptide Fragments; Rabbits; Viral Envelope Proteins

The two envelope proteins of hepatitis C virus, E1 and E2, were expressed in E. coli and, as secretory proteins, in Sf9 insect cells using recombinant baculoviruses. Co-infection of insect cells with E1 and E2-recombinant baculoviruses was performed, which has been shown to result in formation of E1-E2 dimers. All envelope proteins were purified by Ni2+-NTA chromatography and used for screening of serum samples in a HCV EIA assay. Serum samples of normal blood donors, chronically HCV-infected patients, a mixed titer panel and several seroconversion panels were screened and compared to test results with Cobas Core Anti-HCV EIA.. Screening of the sera of chronically HCV-infected patients (100% positive in Cobas Core Anti-HCV EIA) revealed 10-40% anti-E1 positive sera using different Sf9-expressed, glycosylated proteins and 93% using E. coli-expressed, non-glycosylated E1 protein. When the same sera were tested with different E2 proteins expressed in Sf9 cells and in E. coli, about 70-73% showed anti-E2 reactivity. When the proteins from Sf9 cells co-infected with E1- and E2-recombinant baculoviruses were tested, 70-80% of the same sera showed anti-envelope reactivity.. Testing of these patient antisera, and those from the well-characterized mixed titer panel BBI-PHV203, showed that recombinant E1 expressed in E. coli and co-expressed E1 and E2 proteins from Sf9 cells could be used as additional tools for anti-HCV antibody screening.

Topics: Animals; Cell Line; Cloning, Molecular; Dimerization; Escherichia coli; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoenzyme Techniques; Mass Screening; Recombinant Proteins; Reference Values; Reproducibility of Results; Spodoptera; Transfection; Viral Envelope Proteins

Hepatitis C virus (HCV) exists in vivo as a highly variable mixture of closely related genomes (quasispecies), but the pathogenetic significance of such heterogeneity is still largely unknown. To investigate this issue, we compared the composition of HCV quasispecies found in the liver, peripheral blood mononuclear cells (PBMC) and plasma of ten patients by single-strand conformation polymorphism analysis of the E2/NS1 region and sequencing of the variants detected. We found considerable quasispecies differences between the liver and PBMC in all the patients, involving variant numbers, relative quantities and relative electrophoretic mobilities, but no apparent tissue-specific trend. Genome variants present in the liver and/or PBMC were not detected in the corresponding plasma samples, while certain HCV variants were present only in plasma. No dominant amino acids or amino acid pattern characteristic of variants present solely in the PBMC were detected in the E2/NS1 region sequenced.

Topics: Amino Acid Sequence; Base Sequence; DNA, Viral; Genetic Heterogeneity; Hepacivirus; Hepatitis C; Humans; Leukocytes, Mononuclear; Liver; Molecular Sequence Data; Polymorphism, Single-Stranded Conformational; Reproducibility of Results; Viral Envelope Proteins; Viral Nonstructural Proteins

Acute and chronic Hepatitis C virus infections were investigated retrospectively in chimpanzees that had been infected from a single source. Anti-E1 and anti-E2 were detected in two of three chimpanzees with a chronic infection, but were first detected 1 to 2 years after inoculation. Sequence evolution of the E1 region in three animals over a period of 9 to 11 years revealed a mutation rate of 1.02 to 2.23 x 10(-3) base substitutions per site per year. The acute phase viremia levels in acute infections which resolved appeared to be at least 10-fold higher than during the acute phase of chronic infections. During chronic infections, the viral load fell rapidly after the acute phase and remained at very low levels for several years. After 4 to 6 years, the viral load and liver enzymes increased again, suggesting reactivation of the infection. There was no clear temporal relationship between sequence evolution of the E1 region, changes in viral load, and the production of antibodies to the envelope proteins.

Topics: Acute Disease; Animals; Base Sequence; Chronic Disease; Cloning, Molecular; DNA Primers; Evolution, Molecular; Female; Hepacivirus; Hepatitis C; Male; Pan troglodytes; Phylogeny; Polymerase Chain Reaction; RNA, Viral; Viral Envelope Proteins; Viremia

Antibodies directed against hypervariable region 1 (HVR1) within the viral glycoprotein E2 of hepatitis C virus (HCV) are postulated to neutralize virus. An in vitro infection/binding assay of human fibroblast cells was established in order to study neutralization of HCV. Occurrence of mutations in the nucleotide sequence of HVR1 as compared to the inoculum after infection of human fibroblasts suggested replication of HCV in these cells. The significance of HVR1-specific antibodies in sera of patients who were infected in a single-source outbreak by an HCV contaminated anti-D immunoglobulin (IgG) preparation was studied. Using immunoprecipitation and ELISA, HVR1-specific antibodies could be detected in most of the sera obtained early (< or = 1 year p.i.) and late (up to 14 years p.i.) in single patients. Further characterization of the HVR1-specific antibodies in patient sera by attachment studies of HCV to the human fibroblasts suggested that HVR1-specific antibodies in sera obtained early p.i. can neutralize virus of the anti-D IgG preparation.

Topics: Amino Acid Sequence; Antibody Specificity; Base Sequence; Cell Line; Drug Contamination; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoglobulins, Intravenous; Molecular Sequence Data; Neutralization Tests; Precipitin Tests; Viral Envelope Proteins

In patients infected with the hepatitis C virus (HCV), a heterogeneous population of viruses, so-called quasispecies exists in vivo. The hypervariable regions (HVR) within the second envelope gene (HCV-E2) show particularly highly intratypic variability and are considered to be the target of neutralizing antibodies. The aims of the study were to optimize a genotype-independent primer set for amplification of HVR-1 and to establish a sensitive SSCP analysis for rapid and non-isotopic detection of predominant serum HCV quasispecies. Using the optimized SSCP technique, changes of quasispecies composition were investigated in five chronically infected patients with HCV before and during interferon-alpha treatment. HCV genotyping was performed by sequence and phylogenetic analysis. In addition, serial viremia and serum alanine aminotransferase (ALT) levels were determined. The SSCP analysis was performed at two time points before and during interferon-alpha therapy, respectively. Four patients showed an alteration of the SSCP pattern during the first three months of interferon-alpha therapy, whereas in one patient the SSCP pattern changed before therapy and remained stable during treatment with interferon-alpha. The present approach for non-isotopic analysis of single strand conformation polymorphism provides a direct, rapid, and sensitive technique for detection of the heterogeneous population of HCV quasispecies of different genotypes. Using this test procedure, investigations of large cohorts of patients with chronic hepatitis C can be undertaken.

Topics: Alanine Transaminase; Base Sequence; DNA Primers; DNA, Viral; Hepacivirus; Hepatitis C; Humans; Isotope Labeling; Molecular Sequence Data; Polymorphism, Single-Stranded Conformational; Sequence Homology, Nucleic Acid; Viral Envelope Proteins

The envelope protein of hepatitis C virus (HCV) is composed of two membrane-associated glycoproteins, E1 and E2. To obtain HCV E2 protein as a secretory form at a high level, we constructed a recombinant chinese hamster ovary (CHO) cell line expressing a C-terminal truncated E2 (E2t) fused to human growth hormone (hGH), CHO/hGHE2t. The hGHE2t fusion protein was purified from the culture supernatant using anti-hGH mAb affinity chromatography at approximately 80% purity. The purified hGHE2t protein appeared to be assembled into oligomers linked by intermolecular disulfide bond(s) when density gradient centrifugation and SDS-polyacrylamide gel electrophoresis were employed. When the purified fusion protein was used for testing its ability to bind to antibodies specific for HCV by enzyme-linked immunosorbent assay, the protein was recognized by antibodies in sera from 90% of HCV-positive patients. Treatment of hGHE2t protein by beta-mercaptoethanol, but not by heat and SDS, significantly reduced its reactivity to the antibodies of patient sera, suggesting that intermolecular and/or intramolecular disulfide bonds are important for its ability to recognize its specific antibody and that the E2 protein contains discontinuous antigenic epitope(s).

Topics: Animals; Antibodies, Viral; CHO Cells; Cricetinae; Disulfides; Hepacivirus; Hepatitis Antigens; Hepatitis C; Humans; Protein Denaturation; Recombinant Fusion Proteins; Recombinant Proteins; Viral Envelope Proteins

Immunoassays were developed to determine the seroprevalence of antibody against human GB virus C (GBV-C). The antigenic target in each assay was a 44.6-kDa glycosylated protein representing the first 315 amino acids encoded by the GBV-C E2 gene. Sera or plasma were assayed for E2 antibody using an anti-human EIA format in which antigen-coated polystyrene beads were reacted with sample, and bound antibody was detected by addition of enzyme labelled goat anti-human IgG. The presence of anti-E2 antibody was confirmed using a sandwich EIA format in which samples were reacted with antigen coated polystyrene beads, followed by addition of solution phase biotinylated antigen. Detection of antibody captured biotinylated E2 was accomplished by addition of enzyme-conjugated anti-biotin antibody. Antibody against the E2 antigen was detected in 7.4 and 7.8% of 500 sera and 500 plasma, respectively, from US volunteers donating to a Wisconsin blood center, and in approximately 10.7% of hepatitis and retrovirus marker-negative volunteer blood donors from a Missouri blood center. The rate in 1018 sera from US commercial donors at multiple US blood centers was 36.7%. These results indicated a relatively high prevalence of GBV-C exposure in US volunteer donors, and particularly in commercial donors. The clinical implication of the high exposure rate is unclear. These immunoassays are being combined with nucleic acid detection to assess prevalence of GBV-C world wide and to determine if GBV-C plays a role as an etiologic agent.

Topics: Animals; Blood Donors; CHO Cells; Cricetinae; Flaviviridae; Hepacivirus; Hepatitis Antibodies; Hepatitis C; Humans; Immunoassay; Prevalence; Sensitivity and Specificity; Seroepidemiologic Studies; United States; Viral Envelope Proteins

The presence or absence of antibodies to the second envelope protein (anti-E2) of hepatitis C virus (HCV) was determined in stored sera taken from a cohort of 87 Irish women with antibodies to HCV (anti-HCV) who were all infected by HCV genotype 1b from contaminated anti-D immunoglobulin given in 1977. Anti-E2 was found in 16 patients (100%) who were HCV RNA positive but only in 31 of 50 patients (62%) who were HCV antibody positive by recombinant immunoblot assay (RIBA) but HCV RNA negative. In the remaining 21 sera taken from women who had indeterminate recombinant immunoblot assays and who were repeatedly negative on testing for HCV RNA, anti-E2 was found in only three cases (14%). This suggests that loss or absence of anti-E2 may be useful in confirming clearance of HCV.

Topics: Cohort Studies; Female; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Ireland; Viral Envelope Proteins

The frequency of genetic homology of hepatitis C virus (HCV) E2 gene in 8 child-mother pairs was investigated. The route of HCV infection of all children suggested mother-to-child infection by their past history. In this study, we analyzed at least 5 complementary DNA (cDNA) clones for each case. In 6 of the 8 (75%) child-mother pairs, for the region between hypervariable region (HVR)-1 and HVR-2, the similarity of the nucleotide sequence of all 5 cDNA clones between each child and the mother was higher than that between the child and the other children, the other mothers and the sequences from GenBank. One of the 8 (13%) child-mother pairs showed a great similarity in parts of all cDNA clones. This was also confirmed by phylogenetic analyses of the sequences of cDNA clones from families and that from GenBank. These results suggested that the homology search of nucleotide sequence of this region was useful to confirm mother-to-child transmission of HCV.

Topics: Amino Acid Sequence; Child; Child, Preschool; Cloning, Molecular; Female; Gene Frequency; Hepacivirus; Hepatitis C; Humans; Infant; Infant, Newborn; Infectious Disease Transmission, Vertical; Male; Maternal-Fetal Exchange; Molecular Sequence Data; Phylogeny; Pregnancy; Sequence Homology, Amino Acid; Viral Envelope Proteins

The significance of circulating antibody to hepatitis C virus (HCV) envelope glycoprotein 2 (E2)/nonstructural protein 1 (NS1) glycoprotein was studied in 83 patients with chronic HCV infection diagnosed by polymerase chain reaction (PCR). E2/NS1 antibody was quantitatively examined by a passive hemagglutination test using recombinant E2/NS1 glycoprotein encompassing amino acids 388 to 664 of the HCV-H strain. The results were correlated with clinical and virological features such as genotypes and viremic levels assessed by a competitive reverse-transcription PCR assay. E2/NS1 antibody was found in 73 patients (88%), and its occurrence was related to viremic levels. E2/NS1 antibody titers were low in asymptomatic HCV carriers with low levels of viral replication; 9 of 17 such patients tested positive for E2/NS1 antibody (53%), compared with 64 of 66 chronic hepatitis C patients (97%) (P < .01). A significant direct relationship was observed between viremic levels and E2/NS1 antibody titers (r = .52, P < .01). Of the 13 patients with low viremic levels of < 10(6) copies/mL, only 5 tested positive for E2/NS1 antibody (38%), whereas 68 of the 70 patients with viremic levels of > or = 10(6) copies/mL had it (97%) (P < .01). As for the relation to HCV genotypes, no difference was seen in E2/NS1 antibody titers among genotypes examined (1b, 2a, and 2b). These findings suggest that the E2/NS1 antibody tested exhibits no neutralizing activity in chronic HCV infection but may serve as a serological indicator of active virus replication.

Topics: Adult; Aged; Base Sequence; Chronic Disease; Female; Genotype; Hemagglutination Tests; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Viral; Viral Envelope Proteins; Viremia

Hepatitis C virus (HCV) infection is associated with a wide spectrum of liver diseases including cirrhosis and hepatocellular carcinoma (HCC). Although the biological relation between the virus and cirrhosis or HCC is unclear, such variable pathogenicity may be related to the genetic heterogeneity of HCV. Genetic variability of HCV was assessed by determining the nucleotide sequence corresponding to the hypervariable regions (HVR1 and HVR2) of the putative envelope protein (E2/NS1) in positive- and negative-stranded HCV RNA from the cancerous and surrounding non-cancerous liver tissue, peripheral blood mononuclear cells and serum of a patient with HCC. Nineteen distinct HVR1 amino acid sequences (deduced from the nucleotide sequences) were obtained from the patient and could be classified into 5 groups on the basis of the site and time of detection. Some viral isolates with the same HVR1 sequence were shown to replicate in both cancerous and non-cancerous liver tissue, whereas others replicated in HCC tissue only.

Topics: Amino Acid Sequence; Base Sequence; Carcinoma, Hepatocellular; Hepacivirus; Hepatitis C; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Molecular Sequence Data; RNA, Viral; Sequence Analysis; Viral Envelope Proteins

Numerous 2nd and 3rd generation screening and confirmatory assays for the detection of anti-HCV antibodies have been introduced on the international market. The aim of the present study was to compare the performance of five different commercially available screening assays and four 'confirmatory' assays in a panel of serum samples that had tested positive or borderline with a 2nd generation EIA (Abbott HCV EIA 2nd generation). Considerable discrepancies were observed between the different screening assays and confirmatory tests. The antigens from the putative 'core' region of HCV were recognized most frequently by the confirmatory assays. By considering the reactivity to either NS5 (RIBA III and Inno-LIA) or E2/NS1 antigens (Inno-LIA Ab III) no sample could be identified as anti-HCV positive that would otherwise have been regarded as borderline or negative according to its banding pattern with core, NS3 and NS4 proteins. All 24 HCV-RT-PCR positive samples were anti-HCV reactive by the screening EIAs but only 18 and 21 samples were confirmed anti-HCV positive with the RIBA II and III, respectively. A clear association was observed between HCV-RNAemia in serum samples and index values (O.D. sample/O.D. cut-off) of the screening EIAs as well as with the number of reactive proteins in the confirmatory assays. In conclusion, the results of current screening and confirmatory assays are highly divergent. The additional diagnostic significance of the relatively expensive and labour-intensive immunoblots appears to be very limited. For the serological diagnosis of HCV infection and for blood donor screening, confirmatory assays should only be used if there is a borderline result by HCV EIA. The determination of infectivity by qualitative PCR and the follow-up of patients undergoing IFN therapy by HCV-RNA quantification appears to be much more useful.

Topics: Hepacivirus; Hepatitis Antibodies; Hepatitis Antigens; Hepatitis C; Humans; Immunoenzyme Techniques; Reagent Kits, Diagnostic; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins

The use of phylogenetic analyses of partial NS5 and core regions for hepatitis C virus (HCV) genotyping was evaluated by analysing seven Honduran and 24 European HCV strains. Core primers were designed with which HCV genotypes 1, 2, and 3 were readily amplified. The reliability of phylogenetic analysis of a 111-bp core sequence was verified by comparing the typing results with those obtained using the whole core gene of 52 reference strains. Accordant genotypes (1a, 1b, 2b, and 3a) were obtained when phylogenetic analyses were undertaken on both the partial core and a 222-bp NS5 sequence in all of the European HCV strains. Genotypes 1a, 1b, and 3a were identified among the Honduran strains by phylogenetic analysis of the partial NS5 sequence. Interestingly, two of three Honduran type 3a strains, as determined by the NS5 sequence analysis, turned out to be type 1a by core sequence analysis. These two strains were also classified as type 1a, but not 3a, by a core type-specific PCR. Furthermore, the E2/NS1 regions were similar to HCV-PT, a representative strain of genotype 1a. The results indicate that chimeral HCV strains exist, although in most cases a good concordance is found when phylogenetic analysis of partial core and NS5 sequences are used for genotyping. This finding should be taken into account when HCV is genotyped by phylogenetic analysis of a partial HCV sequence from a single genomic region.

Topics: Amino Acid Sequence; Base Sequence; DNA, Viral; Genotype; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Phylogeny; Reproducibility of Results; Sequence Homology, Amino Acid; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins

To determine the clinical significance of viral quasispecies heterogeneity, 59 patients with chronic hepatitis C were studied using singlestranded conformational polymorphism (SSCP) analysis of the HCV E2 hypervariable region 1 (HVR1); of these, 48 were subsequently treated with interferon-alpha. The SSCP method was validated using clones of known nucleotide sequence. HVR1 was amplified in 54 of 59 (92%) patients. The median number of SSCP bands per sample was 6 (range: 2-12). Increased quasispecies heterogeneity correlated with the estimated duration of HCV infection (P < 0.05), parenteral-acquired HCV infection (vs. sporadic, P < 0.05), serum HCV RNA levels (P < 0.05), and HCV genotype 1 infection (P < 0.05), but not with age, serum AST, ALT, or Knodell score. Patients who had complete and sustained response to interferon-alpha (n = 11) had lower pre-treatment quasispecies heterogeneity compared to patients who had complete response with relapse (n = 18, P < 0.05) or no complete response (n = 16, P < 0.01). However, multivariate analysis revealed that HCV viremia was a stronger predictor of response to interferon-alpha. These findings indicate that the estimated duration of HCV carriage, serum HCV RNA levels, and HCV type 1 are important determinants for the evolution of HCV quasispecies heterogeneity; and that increased HCV quasispecies heterogeneity is another marker associated with a poor subsequent response to interferon-alpha.

Topics: Adult; Aged; Chronic Disease; Female; Genetic Heterogeneity; Hepacivirus; Hepatitis C; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Reproducibility of Results; RNA, Viral; Viral Envelope Proteins

Extensive serological testing and HCV RNA determination by RT-PCR was performed in serum, PBMCs, and liver tissue in thirteen anti-HCV reactive patients with persistently normal liver tests. Absolute concordance in the status of HCV RNA between serum, PBMCs, and liver was noted. Five patients were HCV RNA positive but only three had mild histological changes. Eight patients were HCV RNA negative in all three sites and had virtually normal liver histology. Patterns of reactivity in RIBA 2.0 strip immunoblot assay did not differentiate viremic from nonviremic patients. ELISA testing using multiple individual HCV recombinant antigens from the structural and non-structural regions of HCV demonstrated mean antibody titers to the structural antigens, in particular HCV E2 antibodies, to be significantly lower in HCV RNA negative patients. The status of HCV RNA in the serum appears to infer the status of HCV RNA in the liver and PBMCs in patients with persistently normal liver tests. Patients with persistently normal liver tests and undetectable HCV RNA have probably spontaneously cleared HCV infection.

Topics: Adult; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Immunoblotting; Leukocytes, Mononuclear; Liver; Male; Middle Aged; Polymerase Chain Reaction; RNA, Viral; Viral Envelope Proteins

We examined the humoral immune response to recombinant structural proteins of hepatitis C virus (HCV) such as C, E1 and E2 in immunized mice. Mice showed high induction of antibodies against these three structural proteins. Conformational and/or linear epitopes of these regions showed high responses in mice. Comparison with patients revealed higher anti-E1 and anti-E2 responses in mice and 15 immunoreactive peptides which are unique to mice, especially 11 peptides from the E2 region. The hydrophilic regions of these proteins were found to be the most immunogenic. Therefore, the murine immune system against recombinant E1 and E2 glycoproteins was distinct from those of patients in natural infection, and may be a target to find protective activity against HCV infection.

Topics: Animals; Antibody Formation; Base Sequence; Cell Line; Disease Models, Animal; DNA, Viral; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Insecta; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Molecular Sequence Data; Recombinant Fusion Proteins; Viral Core Proteins; Viral Envelope Proteins; Viral Structural Proteins

A systematic virological follow-up of 114 haemodialysis patients treated in the same unit showed that 37, including 17 PCR positive patients, were seropositive for hepatitis C virus (HCV). Type 1b HCV was detected in 10 patients and was much more frequent in this population than in the whole population of patients treated in the hepatogastroenterology departments in southeastern France. The E1/E2 genomic region of seven type 1b HCV strains was sequenced. In four patients, a similar strain was detected in both the E1 variable region and the E2 hypervariable region (HVR1). In addition, two of these four patients were seronegative and PCR negative at the beginning of the study and had not been transfused or transplanted during this period. A phylogenetic tree was drawn which confirmed that these strains were very similar and showed that HCV was transmitted via the nosocomial pathway in this haemodialysis unit.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Base Sequence; Cross Infection; DNA, Viral; Female; Follow-Up Studies; Genotype; Hepacivirus; Hepatitis C; Humans; Male; Middle Aged; Molecular Sequence Data; Phylogeny; Renal Dialysis; Viral Envelope Proteins

The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA. E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1. The medium was concentrated and proteins were purified under native conditions on Ni(2+)-NTA columns. Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase. A isolated from bovine heart and gamma-[32P]ATP. Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients. Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium.

Topics: Adenosine Triphosphate; Animals; Autoradiography; Baculoviridae; Base Sequence; Blotting, Western; Cattle; Cell Line; Cloning, Molecular; Culture Techniques; Cyclic AMP-Dependent Protein Kinases; DNA Primers; Electrophoresis, Polyacrylamide Gel; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Histidine; Humans; Molecular Sequence Data; Myocardium; Phosphorus Radioisotopes; Phosphorylation; Polymerase Chain Reaction; Radioisotope Dilution Technique; Recombinant Proteins; Sequence Tagged Sites; Spodoptera; Transfection; Viral Envelope Proteins

Antibody responses to the hepatitis C virus (HCV) envelope proteins E1 and E2 were analyzed using two original assays in sera from 86 patients in different stages of disease. A Western blot assay and an immunofluorescence assay (IFA) were developed using envelope proteins produced, respectively, in Escherichia coli and in CV1 cells infected with a recombinant SV40. As a third method, the INNO-LIA HCV Ab III assay including E2 synthetic peptides was used. Of 38 chronically infected patients positive for anti-E2 antibodies by IFA, 26 were positive in the Western blot assay (68%) and 25 in the INNO-LIA test (66%). Thus, the detection of anti-envelope antibodies is highly dependent on the antigen formulation, and a native glycosylated form of the proteins is probably needed for their efficient detection. This study shows that the antibody response to HCV envelope proteins depends on the phase of infection. A few acutely infected patients displayed a response to E1 or E2 (36% by Western blot, 7% by IFA), and these antibodies seem to develop in patients evolving toward chronicity. The high prevalence in chronically infected subjects (62% to E2 by Western blot, 90% by IFA), particularly in subjects with essential mixed cryoglobulinemia (68% and 100%), confirms that the resolution of infection involves more than these antibodies. The antienvelope response in patients treated with interferon was investigated, but no significant relationship was found between antibody level prior to treatment and the evolution of hepatitis. The detection of anti-envelope antibodies, therefore, is not predictive of the response to antiviral therapy.

Topics: Acute Disease; Animals; Cell Line; Chlorocebus aethiops; Chronic Disease; Cryoglobulinemia; Gene Expression; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Interferon alpha-2; Interferon-alpha; Recombinant Fusion Proteins; Recombinant Proteins; Viral Envelope Proteins

Some chronic hepatitis C patients show sustained response to interferon (IFN) therapy despite viremia. This condition seems to be related to the density populations of hepatitis C virus (HCV) [Kanto et al. (1995): J Med Virol 46:230-237]. To investigate further the relationship between alanine aminotransferase (ALT) levels after IFN therapy and the HCV density populations, we undertook differential flotation centrifugation of HCV and single strand conformation polymorphism targeted the hypervariable region (HVR) of E2 glycoprotein, which seems to be related to the density populations. Sera were obtained serially from 12 patients who had undergone IFN therapy (six sustained responders with viremia, six nonresponders). During the follow-up after interferon therapy, the HVR heterogeneities changed in 9 of the 12 patients. The remaining three patients whose heterogeneities did not changed persistently showed normal ALT. The changes in HVR heterogeneities were less pronounced in the sustained responders with viremia than in nonresponders; however, their density populations were prominently high in both responders. In two cases, changes in HVR heterogeneities and increase in low-density virion were observed before the hepatitis flare-up. These data indicate that HVR quasispecies show more relation to ALT levels after IFN therapy than HCV density populations and that the changes in the HVR sequences and HCV density populations may be associated with ALT elevation in some patients.

Topics: Adult; Aged; Antiviral Agents; Female; Follow-Up Studies; Hepacivirus; Hepatitis C; Humans; Interferons; Male; Middle Aged; Polymorphism, Single-Stranded Conformational; RNA, Viral; Sensitivity and Specificity; Viral Envelope Proteins

A routine screening test used in the diagnosis of hepatitis C virus (HCV) infection is the anti-HCV antibody (anti-HCV) test containing core, NS3, NS4, and NS5 antigens of HCV. When HCV infection occurs in immunocompromised hosts, antibody formation against core, NS3, or NS4 antigens may be weak in the presence of HCV viremia and cannot be detected by routine anti-HCV tests. This study proposed that in immunocompromised hosts such as patients with chronic renal failure (whose capacity to form antibodies is diminished), antibody formation against the E2 region would be preserved, because the E2/NS1 region of HCV is strongly immunogenic. The aim of this study is to evaluate the significance of anti-E2 in the diagnosis of HCV infection among patients on maintenance hemodialysis who are anti-HCV-negative, using a conventional third-generation enzyme immunoassay (EIA) kit. The E2/NS1 gene of HCV encoding the amino acid sequence 388-664 was molecularly cloned into a vector containing an SV 40 promotor and was expressed in Chinese Hamster ovary cells. Using this E2 protein, the anti-E2 test was performed by EIA on 100 patients on maintenance hemodialysis, and on 50 patients with chronic hepatitis C who were anti-HCV-positive, to evaluate the antigenecity of the E2 protein. Of the 100 hemodialysis patients, 15 (15.0%) tested anti-HCV-positive using a third generation anti-HCV ELISA kit. Of the 85 patients who tested negative for anti-HCV, nine (10.6%) were anti-E2-positive and six (66.7%) of these anti-E2 positive patients showed HCV RNA viremia by HCV reverse transcription-polymerase chain reaction. Fourty-two (84.0%) of 50 patients with chronic hepatitis C were anti-E2-positive. As a control group, we tested for anti-E2 among 30 blood donors who were anti-HCV-negative, and also among 85 patients with hepatocellular carcinoma who were anti-HCV-negative, but in both groups, none (0%) was anti-E2-positive. In conclusion, these data suggest that the E2 protein of HCV should be included in a diagnostic anti-HCV kit for the detection of HCV infection in immunocompromised patients.

Topics: Animals; Blotting, Southern; Chronic Disease; Cricetinae; DNA Probes; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Hepacivirus; Hepatitis Antibodies; Hepatitis C; Humans; Immunoblotting; Immunocompromised Host; Polymerase Chain Reaction; Renal Dialysis; RNA, Viral; Serologic Tests; Viral Envelope Proteins

The clinical significance of the hypervariable region (HVR) in the N-terminus of the E2/NS1 region, which encodes the putative envelope glycoprotein (gp 70) of HCV, has not yet been elucidated. We studied the relation between HVR changes and elevation of the alanine aminotransaminase (ALT) level due to liver cell injury as well as the persistence of HCV infection. Three patients (carrier group) who were HCV RNA positive and had normal ALT levels for as long as five years and three patients with high ALT levels were studied. None of the six patients had a history of treatment. HCV RNA was extracted from serum obtained from each patient in 1990 and 1995. The E2/NS1 region, including HVR-1 and HVR-2, was amplified using the RT-PCR method. PCR products were cloned and nucleotide sequences were determined using the dideoxynucleotide chain termination method. No clear correlation was found between the ALT levels and the number of nucleotide substitutions in HVR-1. The number of nucleotide substitutions in HVR-1 during the five years was greater than in other regions. Furthermore, more nucleotide substitutions occurred in the 1st and 2nd codon positions of HVR-1 than in the control region, even in the carrier group. In conclusion, HVR-1 changes are probably a more important factor in persistent viral infection than liver cell injury.

Topics: Aged; Alanine Transaminase; Amino Acid Sequence; Base Sequence; Carrier State; Codon; Female; Genes, Viral; Hepacivirus; Hepatitis C; Humans; Male; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Viral; Sequence Alignment; Viral Envelope Proteins; Viral Nonstructural Proteins

The detection of antibody to the second envelope protein (E2) of the hepatitis C virus (HCV) has been hampered by the lack of suitable antigens. A previously described E2 recombinant antigen (CHO-E2) expressed as a non-fused, highly glycosylated protein in mammalian cells was used to detect specific antibody (anti-E2) in samples from blood donors and viraemic patients showing positive or indeterminate results for anti-HCV after a wide serological study. Anti-E2 was detected in 50-75% of the donors positive for anti-HCV, 80% of viraemic immunocompetent patients with anti-NS3 alone and 28% of non-viraemic donors with anticore alone. In donors with anti-NS3 (15 samples) or anti-NS4 (51 samples) alone, anti-E2 was found occasionally (3 cases). Moreover, two anti-E2-positive samples from viraemic patients were misidentified by some commercial assays for screening anti-HCV. These results suggest that testing for anti-E2 may be useful for improving the performance of the current assays for anti-HCV screening and confirmation.

Topics: Animals; Antibody Specificity; Blood Donors; CHO Cells; Cricetinae; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Humans; Immunocompetence; Immunocompromised Host; Mass Screening; Middle Aged; Recombinant Proteins; RNA, Viral; Sensitivity and Specificity; Seroepidemiologic Studies; Viral Envelope Proteins; Viremia

The high genetic variability of the 5' end of the envelope protein-coding region E2 (HVR1 E2) of Hepatitis C Virus (HCV) RNA has been suggested by many authors to play an important role in both virus persistence and outcome of liver disease. We studied the relations between HVR1 E2 variability and HCV genotypes, HCV-RNA levels and liver disease in 8 chronic HCV carriers (5 males and 3 females, median age 41 years, followed-up for a mean period of 3 years). Four were healthy HCV carriers with persistently normal ALT levels and normal liver histology and 4 patients with chronic liver disease. In each patient, the HVR1 E2 variability of 2 serum HCV-RNA isolates obtained at least 12 months apart were evaluated by direct sequencing. Nucleotide and amino acid homologies ranged between 97.6%-57.1% and 92.8%-25% in healthy carriers and 95.2%-55.9% and 89.3%-32.1% in patients, respectively. We did not observe any correlation between HVR1 E2 heterogeneity and HCV genotypes, viraemia levels, presence and extent of liver necroinflammation. Our findings suggest that HVR1 E2 heterogeneity has no direct implications in hepatitis, pathogenesis but it could play a major role in virus persistence.

Topics: Adult; Base Sequence; Chronic Disease; DNA Primers; Enzyme-Linked Immunosorbent Assay; Female; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; Retrospective Studies; RNA, Viral; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Viral Envelope Proteins; Viremia

It has been postulated that antibodies specific to the hypervariable region 1 (HVR1) within the putative envelop protein E2 of hepatitis C virus (HCV) can neutralize virus. We studied such antibodies in sera of patients who were infected in a single-source outbreak by a contaminated anti-D immunoglobulin preparation (HCV-AD78). The nucleotide sequences of cDNAs encoding HVR1 of HCV-AD78 were determined. The four major variants (HVR1.A, B, C, and D) were expressed as fusion proteins in Escherichia coli. Sixty-seven percent of sera contained antibodies to HVR1.A. Sera unrelated to infection of the outbreak also recognized HVR1.A but to a lesser extent (15%), suggesting that not all HVR1-specific antibodies are absolutely isolate-specific. Antibodies directed against individual variants of HVR1 were found in sera obtained early postinfection (p.i.) (< or = 1 year) but also in sera obtained several years later. An in vitro binding assay of HCV to tissue culture cells was employed to further characterize these sera. Five of seven sera that were obtained early p.i. prevented binding of HCV to cells. Preincubation of such sera with HVR1-specific fusion proteins restored binding of HCV to cells in four of five sera. These findings suggest that the majority of neutralizing antibodies are directed against HVR1.

Topics: Amino Acid Sequence; Antibody Specificity; Base Sequence; Cells, Cultured; Cloning, Molecular; Disease Outbreaks; Drug Contamination; Epitopes; Escherichia coli; Fibroblasts; Genetic Variation; Hepacivirus; Hepatitis Antibodies; Hepatitis C; Hepatitis C Antibodies; Humans; Molecular Sequence Data; Neutralization Tests; Recombinant Fusion Proteins; Sequence Analysis, DNA; Viral Envelope Proteins

The second envelope protein (E2) of the hepatitis C virus (HCV) was cloned and expressed in Chinese hamster ovary (CHO) cells. This E2 glycoprotein was purified using ion exchange and lectin chromatography and used to construct an enzyme immunoassay for HCV E2 antibodies. The assay was shown to have good specificity, and detection of E2 antibodies was positively correlated (97.3%) to the presence of HCV RNA in serum and plasma. A high concordance between HCV 2.0 and E2 EIA reactivities was also observed. E2 antibody was the first serological marker to appear in 3/5 HCV seroconversion panels. This work demonstrated that 42.4% of core and 15.4% of NS3 indeterminate specimens also contained antibodies to E2, suggesting that HCV infection had occurred in these individuals. The E2 antibody assay was used to evaluate HCV 2.0 EIA-positive, HCV 3.0 EIA-negative plasma donors with indeterminate reactivity on RIBA HCV 2.0 or MATRIX HCV 1.0. Several HCV 3.0-negative specimens were shown to contain E2 antibodies in addition to an original indeterminate serological marker, primarily core. It is concluded that anti-E2 is a useful marker for determining HCV infection, and that the presence of antibodies to two nonoverlapping viral gene products suggests true HCV exposure. New HCV 3.0 blood screening tests should detect HCV 2.0-positive donors who present with an indeterminate pattern by RIBA or MATRIX and who also carry E2 antibodies.

Topics: Animals; Biomarkers; CHO Cells; Cricetinae; Electrophoresis, Polyacrylamide Gel; Hepatitis Antibodies; Hepatitis C; Hepatitis C Antibodies; Humans; Immunoenzyme Techniques; RNA, Viral; Sensitivity and Specificity; Viral Envelope Proteins; Viremia

The physical properties of hepatitis C virus (HCV) particles were determined by ultracentrifugation on 20-60% isopycnic sucrose density gradients. We report that (i) two populations of HCV particles were found in the sera of patients with chronic HCV infection [at high density (1.186-1.213 g/ml) and at low density (1.099-1.127 g/ml)], (ii) virus particles with high density values were associated with immunoglobulin, and (iii) virus particles with low density values accumulated base changes within a hypervariable region (HVR) of the E2 envelope domain of the RNA genome. The results indicate that base changes within the HVR of E2 lead to the accumulation of immunoglobulin-free virus particles. Therefore, these findings imply that persistent HCV infection is established as a consequence of sequence variation in the E2 envelope domain.

Topics: Amino Acid Sequence; Base Sequence; Chronic Disease; DNA, Viral; Hepacivirus; Hepatitis C; Humans; Immunoglobulins; Molecular Sequence Data; RNA, Viral; Ultracentrifugation; Viral Envelope Proteins; Virus Latency

Nucleotide sequences of the hypervariable region (HVR) of the E2/NS1 gene of hepatitis C virus (HCV), which are now thought to contain epitopes for neutralizing antibodies, were compared between antibody-bound HCV and free HCV in a patient with type II cryoglobulinemia. Antibody-bound HCV was immunoprecipitated with anti-human immunoglobulins from serum of the patient. Total RNA was recovered from the pellet and the supernatant, respectively, and the envelope gene containing the HVR was amplified by the reverse transcription and nested polymerase chain reaction. The amplified cDNA was examined by the single strand conformation polymorphism (SSCP) analysis. Sequences of bands separated by SSCP analysis were determined by the dideoxy chain termination method. SSCP analyses revealed that the HCV populations were completely different between antibody-bound HCV and free HCV: antibody-bound HCV was composed of two bands and free HCV was composed of three bands. These five bands showed different mobility with each other on the SSCP gel. Sequencing of each band revealed distinct HVR sequences, differing in 1-34 nucleotides and 1-15 deduced amino acids. Three sequences of free HCV was similar with each other (1-5 nucleotide and 1-4 amino acid differences). On the other hand, two sequences of antibody-bound HCV had 5-34 nucleotide and 5-15 amino acid differences with free HCV. Thirteen amino acids in the 5' of HVR were completely identical in three sequences of free HCV, whereas there were three and seven amino acid differences in two sequences of antibody-bound HCV. These findings suggest that isolated specific epitopes for envelope antibodies exist within the HVR.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Base Sequence; Cryoglobulinemia; DNA Primers; DNA, Viral; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Phylogeny; Polymorphism, Single-Stranded Conformational; Transcription, Genetic; Viral Envelope Proteins

The hepatitis-C virus has been the most prevalent cause of chronic hepatitis in both blood and organ recipients. The introduction of a second-generation immunoassay for antibodies to the hepatitis-C virus (HCV 2.0) provided the opportunity to determine if the hepatitis-C virus can be transmitted through tissue transplantation. Banked sera from tissue donors that had previously been found to be non-reactive to the first-generation hepatitis-C virus antibody assay (HCV 1.0) and non-reactive for antibodies to hepatitis-B core antigen were retested with HCV 2.0. The sera from two donors were reactive; the transplant records of recipients of tissues from these donors were reviewed, and the surgeons or hospitals were contacted. The tissue recipients were tested with HCV 2.0, and positive sera were tested for hepatitis-C virus RNA by polymerase chain reaction. Viral nucleic acids isolated from viremic donors and recipients were analyzed for identity by sequencing of the hepatitis-C virus envelope gene (E2) hypervariable region. There were twenty-one grafts, which had been treated with gamma radiation, from one donor; thirteen had been transplanted to twelve recipients. Serum samples from six of the recipients were tested; one was reactive. This patient had other risk factors for infection with the hepatitis-C virus, and sequence analysis demonstrated non-identity between the donor and recipient hepatitis-C virus isolates. Nine of twelve grafts from a second donor had been transplanted in nine recipients. Serum samples from five patients were tested with HCV 2.0; four were reactive. In three of the four patients, the sera were determined to be positive for the hepatitis-C virus by polymerase chain reaction. E2 sequence analyses of hepatitis-C virus RNA isolates from two of these recipients demonstrated sequence identity with the donor isolate. The results of the present report demonstrate that the hepatitis-C virus can be transmitted by bone, ligament, and tendon allografts. They also support the need for testing of all tissue donors for antibodies to the hepatitis-C virus before the tissue is released for transplantation. The results also suggest that seventeen kilo-gray of gamma radiation may inactivate the hepatitis-C virus in tissue.

Topics: Adolescent; Adult; Aged; Amino Acid Sequence; Base Sequence; Bone Transplantation; Contact Tracing; DNA, Viral; Female; Gamma Rays; Genes, env; Hepacivirus; Hepatitis Antibodies; Hepatitis C; Humans; Immunoblotting; Immunoenzyme Techniques; Male; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; Retrospective Studies; Sequence Analysis, DNA; Tendons; Tissue Transplantation; Viral Envelope Proteins

Hepatitis C virus (HCV) forms complex quasispecies populations which consist of a large number of closely related genetic variants. This genetic heterogeneity may cause antigenic variation or drug resistance. We used heteroduplex analysis by temperature gradient gel electrophoresis (TGGE) to characterize genetic variants of HCV. The high resolution of TGGE was proven by comparison of DNA sequence data of different cDNA clones from the HCV 5'NCR with their corresponding migration pattern in TGGE. Using this method we were able to identify virus variants of the HCV 5'NCR even if they only differed from each other by a single base. HCV populations from three patients with chronic hepatitis C were found to consist of genetic variants, although the degree of the heterogeneity varied. In addition, we compared the genetic heterogeneity of the core and E2 regions of the HCV genome in one patient. Our results demonstrate that TGGE is a useful tool for characterization of the genetic heterogeneity of virus populations in vivo.

Topics: Base Sequence; DNA, Viral; Electrophoresis, Polyacrylamide Gel; Genetic Heterogeneity; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Nucleic Acid Heteroduplexes; Sensitivity and Specificity; Sequence Homology, Nucleic Acid; Temperature; Viral Core Proteins; Viral Envelope Proteins

We report on molecular characterization of hepatitis C virus (HCV) isolates in intravenous drug abusers, as compared to non-drug using patients with posttransfusion hepatitis or sporadic hepatitis of unknown origin. Virus typing was performed by RFLP analysis of PCR products in the 5' NCR. Subtyping was done by hybridization with subtype specific probes or by sequencing in the NS4 and NS5 region, respectively. HCV subtype 1b was found most commonly among all the isolates. However, the subtype 3a had a high prevalence (about 46%) in the group of drug addicts. In these subtype 3a isolates the N-terminal part of the E2 protein was highly variable. This confirms the presence of a hypervariable region (HVR1) in this envelope protein found in all hepatitis C viruses. Each subtype 3a isolate examined had a characteristic unique hypervariable region in the E2 protein. It is noteworthy that there are four amino acids in this region which were highly conserved between all HCV sequences published. It can be assumed that such conserved amino acids are significant for structure and function of this viral protein. In our HCV subtype 3a isolates the NS5 sequences were highly conserved.

Topics: Amino Acid Sequence; Base Sequence; DNA, Complementary; Genetic Variation; Genome, Viral; Genotype; Germany; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Viral; Substance Abuse, Intravenous; Viral Envelope Proteins; Viral Nonstructural Proteins