Composite vector formulation for multiple siRNA delivery as a host targeting antiviral in a cell culture model of hepatitis C virus (HCV) infection.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and cancer worldwide. RNA interference (RNAi)-based gene therapies have emerged recently as a promising tool to treat chronic viral infections. Indeed, small interfering RNAs (siRNAs) provide an opportunity to target host factors required for the viral life cycle. In this study, we evaluated a novel nanovector-based approach for siRNA delivery in a model of chronically infected hepatic cells. We designed original composite nanoparticles by coating the calcium phosphate core with siRNAs targeting HCV host-factors and pyridylthiourea-grafted polyethyleneimine (πPEI). Using combinations of different siRNAs, we observed an efficient and prolonged decrease of HCV replication. Moreover, we showed that the layer-by-layer technique of coating applied to our nanoparticles triggers a sequential release of siRNAs acting on different steps of the HCV life cycle. Together, our results demonstrate the efficacy of these nanoparticles for siRNA delivery and open new perspectives for antiviral therapies.

Lentiviral hepatitis B pseudotype entry requires sodium taurocholate co-transporting polypeptide and additional hepatocyte-specific factors.

Hepatitis B virus (HBV) is one of the world's major unconquered infections, resulting in progressive liver disease, and current treatments rarely cure infection. A limitation to discovering new therapies is our limited knowledge of HBV entry and dissemination pathways that hinders the development of in vitro culture systems. To address this gap in our understanding we optimized the genesis of infectious lentiviral pseudoparticles (HBVpps). The recent discovery that the bile salt transporter sodium taurocholate co-transporting polypeptide (NTCP) acts as a receptor for HBV enabled us to assess the receptor dependency of HBVpp infection. HBVpps preferentially infect hepatoma cells expressing NTCP, whereas other non-liver cells engineered to express NTCP do not support infection, suggesting that additional hepatocyte-specific factors are required for HBVpp internalization. These results highlight the value of the HBVpp system to dissect the pathways of HBV entry and dissemination.

Prevention of hepatitis C virus infection by adoptive allogeneic immunotherapy using suicide gene-modified lymphocytes: an in vitro proof-of-concept.

Hepatitis C virus (HCV)-induced, end-stage liver disease is a major indication for liver transplantation, but systematic graft reinfection accelerates liver disease recurrence. Transplantation recipients may be ineligible for direct-acting antivirals, owing to toxicity, resistance or advanced liver disease. Adoptive immunotherapy with liver graft-derived, ex vivo-activated lymphocytes was previously shown to prevent HCV-induced graft reinfections. Alternatively, the applicability and therapeutic efficacy of adoptive immunotherapy may be enhanced by 'ready for use' suicide gene-modified lymphocytes from healthy blood donors; moreover, conditional, prodrug-induced cell suicide may prevent potential side effects. Here, we demonstrate that allogeneic suicide gene-modified lymphocytes (SGMLs) could potently, dose- and time-dependently, inhibit viral replication. The effect occurs at effector:target cell ratios that exhibits no concomitant cytotoxicity toward virus-infected target cells. The effect, mediated mostly by CD56+ lymphocytes, is interleukin-2-dependent, IFN-γ-mediated and, importantly, resistant to calcineurin inhibitors. Thus, post-transplant immunosuppression may not interfere with this adoptive cell immunotherapy approach. Furthermore, these cells are indeed amenable to conditional cell suicide; in particular, the inducible caspase 9 suicide gene is superior to the herpes simplex virus thymidine kinase suicide gene. Our data provide in vitro proof-of-concept that allogeneic, third-party, SGMLs may prevent HCV-induced liver graft reinfection.

New aspects of an anti-tumour drug: sorafenib efficiently inhibits HCV replication.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and is associated with significant morbidity and mortality. Since there is evidence for an interaction of NS5A with c-Raf we studied whether the c-Raf inhibitor sorafenib affects HCV replication. METHODS: HCV replicating HuH7.5 cells were treated with sorafenib and examined for HCV RNA titres by northern blotting or real time polymerase chain reaction (PCR), for core, NS3 and NS5A expression by immunostaining, and for replication by luciferase reporter assays. RESULTS: Here we demonstrate that in cells replicating infectious HCV particles, NS5A recruits c-Raf to the replicon complex resulting in the activation of c-Raf. Therefore, we studied the effect of inhibition of c-Raf on HCV replication using the anti-tumour drug sorafenib that is known to inhibit c-Raf with high specificity. Sorafenib efficiently blocks HCV replication and viral gene expression. In addition, in HCV-replicating cells sorafenib decreased the hyperphosphorylated form of NS5A and resulted in the formation of additional hypophosphorylated forms. Further, sorafenib caused a rapid dissociation of lipid droplets. We provide evidence that the antiviral effect of sorafenib indeed is caused by inhibition of c-Raf. By contrast, inhibition of targets downstream of c-Raf or inhibition of tyrosine kinases by sunitinib did not affect HCV replication. CONCLUSION: Our data demonstrate that the well-characterised anti-tumour drug sorafenib efficiently blocks HCV replication in vitro. This novel effect of sorafenib should be further explored as an antiviral strategy for patients with chronic HCV infection.

[Study of hepatitis C virus leukotropism by characterization of viral quasispecies in the liver transplantation setting]. / Etude du leucotropisme du virus de l'hépatite C par analyse des quasi-espèces virales dans le contexte de la transplantation hépatique.

Besides hepatocytes, representing the main replication site of hepatitis C virus, peripheral blood mononuclear cells also represent a crucial target for viral infection. Hepatitis C virus compartmentalization (i.e., non-random distribution) of viral variants between plasma and peripheral blood mononuclear cells, more frequently observed in liver transplant patients compared to non-transplanted patients, makes liver transplantation an interesting model for the analysis of hepatitis C leukotropism. This article aims to present, firstly, in clinical and biological features arguing favour of hepatitis C virus infection leukotropism and, secondly, to review current knowledge about compartmentalization between plasma and peripheral blood mononuclear cells, especially in the liver transplantation setting.

Genetic variants of hepatitis B virus and their clinical relevance.

Infection with hepatitis B virus (HBV) leads to a wide spectrum of clinical presentations ranging from an asymptomatic carrier state to self-limited acute or fulminant hepatitis to chronic hepatitis with progression to cirrhosis and hepatocellular carcinoma (HCC). Infection with HBV is one of the most common viral diseases affecting man. Both viral factors as well as the host immune response have been implicated in the pathogenesis and clinical outcome of HBV infection. Evidence has been accumulating that HBV mutants are associated with certain clinical disease manifestations, may affect the natural course of the infection and confer resistance to antivirals. Naturally occurring mutations have been identified in the structural and non-structural genes as well as regulatory elements of the virus. The best characterized mutants comprise the pre-core (pre-C) stop codon mutation resulting in a loss of hepatitis B e antigen (HBeAg), defined clusters of mutations in the core promotor resulting in enhanced viral replication and mutations in the hepatitis B core and surface antigens (HBcAg and HBsAg) altering the antigenicity of the virus. More recently, several mutations in the reverse transcriptase/polymerase gene have been identified conferring resistance to antivirals used for the treatment of chronic hepatitis B. In this review, we will focus on the biological phenotype of HBV genetic variants and discuss their clinical relevance for the pathogenesis of HBV-induced liver disease.

Induction of hepatitis C virus E1 envelope protein-specific immune response can be enhanced by mutation of N-glycosylation sites.

Deglycosylation of viral glycoproteins has been shown to influence the number of available epitopes and to modulate immune recognition of antigens. We investigated the role played by N-glycans in the immunogenicity of hepatitis C virus (HCV) E1 envelope glycoprotein, a naturally poor immunogen. Eight plasmids were engineered, encoding E1 protein mutants in which the four N-linked glycosylation sites of the protein were mutated separately or in combination. In vitro expression studies showed an influence of N-linked glycosylation on expression efficiency, instability, and/or secretion of the mutated proteins. Immunogenicity of the E1 mutants was studied in BALB/c mice following intramuscular and intraepidermal injection of the plasmids. Whereas some mutations had no or only minor effects on the antibody titers induced, mutation of the fourth glycosylation site (N4) significantly enhanced the anti-E1 humoral response in terms of both seroconversion rates and antibody titers. Moreover, antibody induced by the N4 mutant was able to recognize HCV-like particles with higher titers than those induced by the wild-type construct. Epitope mapping indicated that the E1 mutant antigens induced antibody directed at two major domains: one, located at amino acids (aa) 313 to 332, which is known to be reactive with sera from HCV patients, and a second one, located in the N-terminal domain of E1 (aa 192 to 226). Analysis of the induced immune cellular response confirmed the induction of gamma interferon-producing cells by all mutants, albeit to different levels. These results show that N-linked glycosylation can limit the antibody response to the HCV E1 protein and reveal a potential vaccine candidate with enhanced immunogenicity.

Hepatitis C virus-like particles induce virus-specific humoral and cellular immune responses in mice.

We have recently described the production of hepatitis C virus-like particles (HCV-LPs) in insect cells that resemble the putative virions. Here we evaluate the humoral and cellular immunogenicity of the virus-like particles with or without viral p7 protein, a small viral polypeptide that resides between the structural and nonstructural regions of the HCV polyprotein and whose function has not been defined. Immunized BALB/c mice developed high titers of anti-E2 antibodies and virus-specific cellular immune responses including cytotoxic T lymphocytes and T helper responses with gamma interferon production. The virus-like particles without p7 generated a higher cellular immune response with a more T(H)1 profile than the particles with p7. Immunization of heat-denatured particles resulted in substantially lower humoral and cellular responses, suggesting that the immunogenicity is strongly dependent on particle formation. Administration of CpG oligonucleotide or cationic lipid 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), two potent adjuvants, did not significantly enhance the immunogenicity of HCV-LPs. Our results indicate that HCV-LPs can induce humoral and cellular immune responses and offer a promising approach to vaccine development.

Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate.

BACKGROUND & AIMS: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. Successful vaccine development is crucial in controlling global HCV infection. We have previously described the generation of HCV-like particles (HCV-LPs) in insect cells using a recombinant baculovirus containing the complementary DNA of the HCV structural proteins. These HCV-LPs had similar morphological and biophysical properties as the putative virions. In this study, we analyzed the structural features, antigenic composition, seroreactivity, and immunogenicity of purified HCV-LPs. METHODS: HCV-LPs were analyzed by electron microscopy and antibody immunolabeling and precipitation. An enzyme-linked immunosorbent assay (ELISA) using HCV-LPs was developed. The humoral response to HCV-LPs in mice was studies by core and envelope ELISAs, Western immunoblotting, and immunofluorescence. RESULTS: Structural and antigenic compositions of HCV-LPs were shown to be similar to those of putative HCV virions. Using the HCV-LP ELISA, high-titer anti-HCV antibodies were detected in individuals infected with various HCV genotypes. In vivo, HCV-LPs elicited a humoral response broadly directed against HCV structural proteins. CONCLUSIONS: HCV-LPs resemble HCV virions and are capable of inducing a humoral response targeted against various regions of HCV structural proteins, suggesting that HCV-LPs may be promising as a potential vaccine candidate.

Naturally occurring mutations define a novel function of the hepatitis B virus core promoter in core protein expression.

Functional analysis of naturally occurring hepatitis B virus (HBV) mutations is crucial in understanding their impact on disease. We have recently identified two mutations in the HBV core promoter of an HBV strain associated with fulminant hepatitis leading to highly (15-fold) enhanced replication as a result of increased viral encapsidation of pregenomic RNA into the core particles (T. F. Baumert et al., J. Clin. Invest. 98:2268-2276, 1996). Functional studies in an encapsidation assay had demonstrated that the increase in encapsidation was largely independent of pregenomic RNA transcription. In this study, we define the molecular mechanism whereby the two core promoter mutations (C to T at nucleotide [nt] 1768 and T to A at nt 1770) result in enhanced viral encapsidation and replication. The effect of these mutations leading to increased encapsidation is mediated through enhanced core protein synthesis (15-fold) by the mutant virus. The marked increase in core protein synthesis is largely a result of posttranscriptional or translational effect of the mutations because the mutations resulted in only a twofold increase in pregenomic RNA transcription. In addition, this effect appears to be selective for core expression since reverse transcriptase-polymerase expression was increased only twofold. trans-complementation analyses of HBV replication demonstrated that enhanced replication occurred only when the mutations were provided together with the core protein in trans, confirming the functional association of the core promoter mutations and core protein expression. In addition, the effect of the mutations appears to be quantitatively dependent on the strain background to which the mutations were introduced. Our study suggests that the HBV core promoter regulates core protein expression at both transcriptional and posttranscriptional levels.

Hepatitis C virus structural proteins assemble into viruslike particles in insect cells.

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of HCV has been hampered by the low level of viral particles in infected individuals, the inability to propagate efficiently the virus in cultured cells, and the lack of a convenient animal model. Due to these obstacles, neither the structure of the virus nor the prerequisites for its assembly have been clearly defined. In this report, we describe a model for the production and purification of HCV-like particles in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural proteins. In insect cells, expressed HCV structural proteins assembled into enveloped viruslike particles (40 to 60 nm in diameter) in large cytoplasmic cisternae, presumably derived from the endoplasmic reticulum. Biophysical characterization of viruslike particles by CsCl and sucrose gradient centrifugation revealed biophysical properties similar to those of putative virions isolated from infected humans. The results suggested that HCV core and envelope proteins without p7 were sufficient for viral particle formation. Analysis of particle-associated nucleic acids demonstrated that HCV RNAs were selectively incorporated into the particles over non-HCV transcripts. The synthesis of HCV-like particles in insect cells may provide an important tool to determine the structural requirements for HCV particle assembly as well as to study viral genome encapsidation and virus-host interactions. The described system may also represent a potential approach toward vaccine development.

Two core promotor mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication.

Viral mutations have been implicated in alteration of the biological phenotype of hepatitis B virus (HBV). We recently cloned and sequenced the viral genome of an HBV strain associated with an outbreak of fulminant hepatitis (FH strain). The FH strain contained numerous mutations in all genomic regions and was functionally characterized by a more efficient encapsidation of pregenomic RNA leading to highly enhanced replication. To define the responsible mutation(s) for the enhanced replication, we introduced individual mutations of the FH strain into a wild-type construct by oligonucleotide-directed mutagenesis. Analysis of viral replication showed that two adjacent mutations in the HBV core promotor (C to T at nucleotide 1768 and T to A at nucleotide 1770) led to high level replication. Similar to the FH strain, this mutant displayed the phenotype of enhanced encapsidation of pregenomic RNA. Functional studies in an encapsidation assay demonstrated that the identified mutations resulted in a minor increase of pregenomic RNA transcription (two- to threefold) and a major transcription-independent enhancement (> 10-fold) of viral encapsidation. Our results demonstrate that the two adjacent mutations in the HBV core promotor region are responsible for the enhanced replication of the FH strain. These two mutations, outside the previously described encapsidation signal, core, and polymerase polypeptides, appeared to affect a novel genetic element involved in viral encapsidation.