In vitro assembly of nucleocapsid-like particles from purified recombinant capsid protein of dengue-2 virus.

The capsid protein is one of the three structural proteins of flaviviruses and is the building block of the nucleocapsid. It has also a predominant role in the replication of dengue virus. To obtain nucleocapsid-like particles from recombinant dengue-2 capsid protein produced in E. coli, a purification process using cation exchange chromatography was established. The purified protein exhibited a molecular mass corresponding to a dimer; therefore, similar to that reported for alphaviruses, an in vitro assembly reaction using single-stranded DNA was performed. In all cases, particles were obtained independently of the specificity and the length of the oligonucleotides used. The present work is the first report of in vitro assembly of the recombinant dengue capsid protein, which could constitute a powerful tool in the development of vaccine candidates.

Preparation of plasmid DNA-containing liposomes using a high-pressure homogenization--extrusion technique.

High-pressure homogenization-extrusion (HPHE) is a method that can be used for downsizing large lipid vesicles with commercially available instrumentation (e.g., from Avestin Inc., Canada), which covers a full range of processing capacities from laboratory (0.5-3.5 mL) to large-scale continuous (1-1000 L/h) production. Consequently, the feasibility (at the laboratory scale) of using HPHE for producing DNA-loaded liposomes by the conventional dehydration-rehydration method was explored. HPHE-generated small unilamellar vesicles had a mean size in the range of 27-76 nm depending on the number of processing cycles and lipid (PC:DOPE:DOTAP or PC:DOPE:Ethyl-DOPC, 1:0.5:0.5, mol/mol) formulation. The size could be further regulated by the pore size (50 or 100 nm) of the extrusion membrane. Using plasmids for the V3 loop of HIV-1, and the capsid, E1 and E2 of hepatitis C, entrapment yields of 72-98.2% into dehydrated-rehydrated vesicles (DRV) were obtained over a wide range (0.309-2.5 mg) of DNA quantities. Most of the plasmid DNA was retained by liposomes even in the presence of sodium dodecyl sulfate (from 0.05% to 0.3%) and efficiently protected from nuclease-mediated degradation. Although the encapsulation process slightly decreased (in the range of 42.8-65.7%) the relative abundance of plasmid super coiled isoforms, the transfection efficiency of monkey kidney COS-7 cells with the plasmid DNA extracted from liposomes (9+/-0.4%) was similar to that of the non-treated DNA (8.7+/-0.2%), using the commercial SuperFect(R) Transfection Reagent. Also, it was found that an appreciable loss of lipid mass-either associated with the HPHE or the dehydration-rehydration steps-occurs during the liposome manufacturing process. These results at the bench scale are a useful reference for planning pilot or large-scale manufacture of DNA vaccine-containing liposomes.

HCV core protein localizes in the nuclei of nonparenchymal liver cells from chronically HCV-infected patients.

Understanding the mechanism of hepatitis C virus (HCV) pathogenesis is an important part of HCV research. Recent experimental evidence suggests that the HCV core protein (HCcAg) has numerous functional activities. These properties suggest that HCcAg, in concert with cellular factors, may contribute to pathogenesis during persistent HCV infection. HCV is capable of infecting cells other than hepatocytes. Although the extrahepatic cellular tropism of HCV may play a role in the pathophysiology of this infection, the precise biological significance of the presence of HCV components in different liver cell types presently remains to be established. In this study, HCcAg was detected in nonparenchymal liver cells of six patients out of eight positive for serum HCV RNA. Immunostaining with anti-HCcAg mAbs revealed the presence of this protein in different liver cell types such as lymphocytes, Kupffer, polymorphonuclear, pit, endothelial, stellate, and fibroblast-like cells. Interestingly, HCcAg was immunolabeled not only in the cytoplasm but also in the nucleus of these cells. Remarkably, HCcAg co-localized with large lipid droplets present in stellate cells and with collagen fibers in the extracellular matrix. Moreover, HCcAg was immunolabeled in bile canaliculus suggesting the involvement of the biliary system in the pathobiology of HCV. Data suggest that nonparenchymal liver cells may constitute a reservoir for HCV replication. Besides, HCcAg may contribute to modulate immune function and fibrosis in the liver as well as steatosis.

In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein.

Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.

Ultrastructural evidences of HCV infection in hepatocytes of chronically HCV-infected patients.

In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied by transmission electron microscopy (EM) and immunoelectron microscopy (IEM). The 13 biopsies showed ultrastructural cell damage typical of acute viral hepatitis. In four of the 13 liver biopsies enveloped virus-like particles (VLPs) inside cytoplasmic vesicles and in the cytoplasm of hepatocytes were observed. We also detected the presence of unenveloped VLPs mainly in the cytoplasm and in the endoplasmic reticulum. IEM using anti-core, E1 and E2 monoclonal antibodies (mAbs) confirmed the specific localization of these proteins, in vivo, inside cytoplasm and endoplasmic reticulum. Thus, this work provided evidence for hepatocellular injury related to HCV infection. It also suggested the presence of HCV-related replicating structures in the cytoplasm of hepatocytes and raised the possibility of hepatitis C virion morphogenesis in intracellular vesicles.