The Hepatitis C Virus Glycan Shield and Evasion of the Humoral Immune Response

Despite the induction of effective immune responses, 80% of hepatitis C virus (HCV)-infected individuals progress from acute to chronic hepatitis. In contrast to the cellular immune response, the role of the humoral immune response in HCV clearance is still subject to debate. Indeed, HCV escapes neutralizing antibodies in chronically infected patients and reinfection has been described in human and chimpanzee. Studies of antibody-mediated HCV neutralization have long been hampered by the lack of cell-culture-derived virus and the absence of a small animal model. However, the development of surrogate models and recent progress in HCV propagation in vitro now enable robust neutralization assays to be performed. These advances are beginning to shed some light on the mechanisms of HCV neutralization. This review summarizes the current state of knowledge of the viral targets of anti-HCV-neutralizing antibodies and the mechanisms that enable HCV to evade the humoral immune response. The recent description of the HCV glycan shield that reduces the immunogenicity of envelope proteins and masks conserved neutralizing epitopes at their surface constitutes the major focus of this review

Enhanced anti-HCV activity of interferon alpha 17 subtype

<p>Abstract</p> <p>Background</p> <p>Pegylated interferon alpha 2 (a or b) plus ribavirin is the most effective treatment of chronic hepatitis C but a large proportion of patients do not respond to therapy. So, it is interesting to improve the treatment efficacy. Interferon alpha is a type I interferon composed of 12 different subtypes. Each subtype signals by the Jak-Stat pathway but modulations in the antiviral activity was previously described.</p> <p>Methods</p> <p>Using the hepatitis C virus (HCV) culture system, we have tested the anti-HCV activity of each interferon alpha subtypes. We have analyzed the effect of each subtype on the HCV multiplication and the cell-signaling pathway for some subtypes.</p> <p>Results</p> <p>There were divergent effects of IFN alpha subtypes against HCV. We have found that IFN alpha 17 was three times more efficient than IFN alpha 2a on HCV. This efficiency was related to a stronger stimulation of the Jak-Stat pathway.</p> <p>Conclusion</p> <p>We suggest that IFN α17 should be tested therapeutically with a view to improving treatment efficacy.</p

A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

BACKGROUND/AIM: The role of humoral immunity in hepatitis C virus (HCV) infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. METHODS: The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. RESULTS: The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively). The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134) for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85) for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles system. CONCLUSION: This study presents a simple, specific and reproducible cell culture-based assay for determination of HCV-neutralizing antibodies in human sera. The assay should be an important tool for gauging the relationship between the neutralizing antibodies response and viral load kinetics in acutely or chronically infected patients and for investigating the possible eradication or prevention of HCV infection by neutralizing antibodies

Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly

International audienceHepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis

The treatment response of chronically hepatitis C virus-infected patients depends on interferon concentration but not on interferon gene expression in peripheral blood mononuclear cells.

International audienceThe current treatment of chronic hepatitis C is based on pegylated alpha interferon (PEG-IFN-α) and ribavirin. The aim of this study was to identify biological and clinical variables related to IFN therapy that could predict patient outcome. The study enrolled 47 patients treated with PEG-IFN and ribavirin combined therapy. The interferon concentration was measured in serum by a bioassay. The expression of 93 interferon-regulated genes in peripheral blood mononuclear cells was quantified by real-time quantitative reverse transcription-PCR (RT-PCR) before and after 1 month of treatment. The interferon concentration in the serum was significantly lower in nonresponders than in sustained virological responders. Moreover, a significant correlation was identified between interferon concentration and interferon exposition as well as body weight. The analysis of interferon-inducible genes in peripheral blood mononuclear cells among the genes tested did not permit the prediction of treatment outcome. In conclusion, the better option seems to be to treat patients with weight-adjusted PEG-IFN doses, particularly for patients with high weight who are treated with PEG-IFN-α2a. Although the peripheral blood mononuclear cell samples are the easiest to obtain, the measurement of interferon-inducible genes seems not be the best strategy to predict treatment outcome

Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codonswitching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic "fossil record" of historical purifying selection against E1E2 intermediate phenotypes

NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins towards Virus Assembly

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly

Étiologie virale du diabète de type I

Le diabète de type 1 est une maladie connue depuis plusieurs milliers d années, caractérisé par un dysfonctionnement du métabolisme glucidique dû à une destruction des cellules b du pancréas. C est une maladie chronique d étiologie multifactorielle. Bien que de multiples recherches aient été entreprises depuis le début du siècle, la cause exacte n est pas encore définie à l heure actuelle, même si l intervention d un facteur héréditaire est aujourd hui bien établie. Il ne s agit pas d un gène de maladie, mais d un terrain de susceptibilité , sans doute polygénique qui constitue la condition préalable favorisant l expression de la maladie avec le concours probable de facteurs environnementaux. Parmi ceux-ci, on pourra citer : l allaitement, le gluten, une alimentation riche en nitrosamines, un environnement froid, le stress ou encore l hygiène Mais, le premier et le plus incriminé des facteurs environnementaux reste l exposition à certains virus tels le virus de la rubéole ou encore le virus Coxsackie B. Le mécanisme le plus probable est le déclenchement d un processus auto-immun par l infection virale, ayant pour résultat une destruction secondaire de la cellule b sur une période plus ou moins longue, tout en sachant que la contribution relative des différents éléments génétiques, immunitaires et environnementaux à l étiopathogénie est sans doute variable d un cas à l autre.AMIENS-BU Santé (800212102) / SudocSudocFranceF

Le virus du chikungunya (évolution et perspectives)

Le chikungunya est un arbovirus transmis par les moustiques du genre Aedes qui sévit sur un mode épidémique et endémique. C est une maladie tropicale d apparition brutale ressemblant fortement à la dengue. Les symptômes les plus fréquents sont la fièvre, les éruptions cutanées et les arthralgies. La prise en charge est habituellement simple avec un traitement symptomatique car aucun traitement antiviral n existe. La prévention est le meilleur moyen d enrayer une épidémie et elle repose sur des mesures individuelles et collectives. Aucun vaccin n est actuellement disponible. Le chikungunya ne touchait que l Afrique, l Asie et le sous continent indien jusqu à l épidémie de 2005 survenant sur les îles de l Océan Indien avec pour cible principale La Réunion. Cette épidémie était la première à survenir dans un pays développé et a en outre surpris par sa magnitude et la survenue de formes graves voire mortelles. Les autorités de santé restent vigilantes car l Europe a déjà subi la propagation du virus : une épidémie est survenue dans le Nord de l Italie en juin 2005. Le virus n a pas sévi sur le continent européen depuis cet incident mais les échanges internationaux pourraient favoriser la survenue d une épidémie dans d autres pays où le vecteur est présent. Le pourtour méditerranéen de la France en est l exemple type : le vecteur Aedes albopictus y est présent par endroits depuis 4 ans et la région est pourvue d un climat similaire à la région italienne où l épidémie est survenue. Les plans de prévention sont donc renforcés et la surveillance y est accrue. Les pharmaciens d officine sont au premier plan sur les conseils en matière de préventionAMIENS-BU Santé (800212102) / SudocSudocFranceF