Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of viral entry and infection has been hampered by the inability to efficiently propagate the virus in cultured cells and the lack of a small-animal model. Recent studies have shown that in insect cells, the HCV structural proteins assemble into HCV-like particles (HCV-LPs) with morphological, biophysical, and antigenic properties similar to those of putative virions isolated from HCV-infected humans. In this study, we used HCV-LPs derived from infectious clone H77C as a tool to examine virus-cell interactions. The binding of partially purified particles to human cell lines was analyzed by fluorescence-activated cell sorting with defined monoclonal antibodies to envelope glycoprotein E2. HCV-LPs demonstrated dose-dependent and saturable binding to defined human lymphoma and hepatoma cell lines but not to mouse cell lines. Binding could be inhibited by monoclonal anti-E2 antibodies, indicating that the HCV-LP-cell interaction was mediated by envelope glycoprotein E2. Binding appeared to be CD81 independent and did not correlate with low-density lipoprotein receptor expression. Heat denaturation of HCV-LPs drastically reduced binding, indicating that the interaction of HCV-LPs with target cells was dependent on the proper conformation of the particles. In conclusion, our data demonstrate that insect cell-derived HCV-LPs bind specifically to defined human cell lines. Since the envelope proteins of HCV-LPs are presumably presented in a virion-like conformation, the binding of HCV-LPs to target cells may allow the study of virus-host cell interactions, including the isolation of HCV receptor candidates and antibody-mediated neutralization of binding.Hepatitis C virus (HCV) is a major cause of posttransfusion and community-acquired hepatitis (2, 3, 13, 34). The majority of HCV-infected individuals develop chronic hepatitis that may progress to liver cirrhosis and hepatocellular carcinoma (34, 46). Treatment options for chronic HCV infection are limited, and a vaccine to prevent HCV infection is not available (31,33,34).HCV has been tentatively classified in a separate genus (Hepaticivirus) of the family Flaviviridae (4, 36, 43). The virion contains a positive-stranded RNA genome of approximately 9.6 kb. The genome consists of a highly conserved 5Ј noncoding region followed by a long open reading frame of 9,030 to 9,099 nucleotides (nt) that is translated into a single polyprotein of 3,010 to 3030 amino acids (aa) (4, 36). Processing of the polyprotein occurs with a combination of host and viral proteases. The HCV structural proteins comprise the putative nucleocapsid or core protein and the two envelope glycoproteins, E1 and E2 (4, 36, 43). The cleavage of structural proteins from the polyprotein is catalyzed by a host signal peptidase. Envelope proteins E1 and E2 are transmembrane proteins consisting of a large N-terminal ectodomain and a C-terminal hydrophobic anchor. E1 and E2 are posttranslationally modified by extensive N-linke...
Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis worldwide. 1-5 Most HCV-infected individuals develop chronic hepatitis progressing eventually to cirrhosis and hepatocellular carcinoma. 6 In the United States, 2.7 million persons are chronically infected with HCV. 7 Drug use and high-risk sexual behavior have been identified as major risk factors for HCV infection. 7 Treatment options for chronic HCV infection are limited. 1,[8][9][10][11] Although the humoral and cellular immune responses induced by HCV have been described in great detail, mechanisms of viral clearance and persistence are still poorly understood. Chronic HCV infection results in the induction of a strong humoral immune response, 1,12 and anti-HCV antibodies can be detected easily using synthetic peptides or recombinant proteins in serologic assays (for review, see Schiff 13 ). Because these antigens are either synthetic peptides or recombinant truncated proteins representing mostly linear epitopes, little information is available regarding anti-HCV antibodies against nonlinear or conformational epitopes and their relevance in the pathogenesis of hepatitis C.HCV is a member of the Flaviviridae family. 4 The virion contains a positive-stranded RNA genome that is translated into a single polyprotein. This polyprotein is processed by host and viral proteases. 5,14 The HCV structural proteins comprise the core protein (C) and the 2 envelope glycoproteins, E1 and E2. We have recently described the synthesis of HCVlike particles (HCV-LPs) in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural protein core, E1, and E2. 15 The insect cell-derived HCV-LPs exhibit similar morphologic and biophysical properties as putative virions isolated from HCV-infected humans. Because HCV-LPs synthesized in insect cells are derived from partial viral genome without the nonstructural genes required for viral replication, they are noninfectious and therefore represent an attractive candidate for HCV vaccine. 16 In contrast to previously described serologic assays, the HCV proteins of HCV-like particles are presumably presented in a native, virion-like conformation, and may therefore interact with anti-HCV antibodies directed against nonlinear or conformational epitopes of HCV envelope proteins that may represent neutralizing epitopes.Abbreviations: HCV, hepatitis C virus; HCV-LP, hepatitis C virus-like particle; E1/E2, envelope glycoproteins 1 and 2; ELISA, enzyme-linked immunosorbent assay; IFN-␣, interferon alfa; ALT, alanine transaminase; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; OD, optical density; SR/NR, sustained responders/nonresponders; Rel, relapsers.From the
The pathological role of ApoE4 in Alzheimer's disease (AD) is not fully elucidated yet but there is strong evidence that ApoE is involved in Abeta deposition, which is an early hallmark of AD neuropathology. Overexpression of ApoE in neuroblastoma cells (Neuro2a) leads to the generation of an intracellular 13 kDa carboxy-terminal fragment of ApoE comparable to fragments seen in brains of AD patients. ApoE4 generates more of this fragment than ApoE2 and E3 suggesting a potential pathological role of these fragments in Alzheimer's disease. Analysis of this intracellular ApoE4 fragment by protease digest followed by MALDI-TOF mass spectrometry showed the proteolytic cleavage site close to residue 187 of ApoE. We have engineered and expressed the corresponding ApoE fragments in vitro. The recombinant 13 kDa carboxy-terminal fragment inhibited fibril formation of Abeta; this contrasts with the full-length ApoE and the corresponding amino-terminal ApoE fragment. Moreover, we show that the 13 kDa carboxy-terminal fragment of ApoE stabilizes the formation of Abeta hexamers. Complexes of Abeta with the 13 kDa carboxy-terminal ApoE fragment show toxicity in PC12 cells comparable to Abeta fibrils. These data suggest that cleavage of ApoE, leading to the generation of this fragment, contributes to the pathogenic effect of ApoE4 in AD.
The development of a vaccine to prevent congenital human cytomegalovirus (HCMV) disease is a public health priority. We tested rhesus CMV (RhCMV) prototypes of HCMV vaccine candidates in a seronegative macaque oral challenge model. Immunogens included a recombinant pentameric complex (PC; gH/gL/pUL128/pUL130/pUL131A), a postfusion gB ectodomain, and a DNA plasmid that encodes pp65-2. Immunization with QS21-adjuvanted PC alone or with the other immunogens elicited neutralizing titers comparable to those elicited by RhCMV infection. Similarly, immunization with all three immunogens elicited pp65-specific cytotoxic T cell responses comparable to those elicited by RhCMV infection. RhCMV readily infected immunized animals and was detected in saliva, blood and urine after challenge in quantities similar to those in placebo-immunized animals. If HCMV evades vaccine-elicited immunity in humans as RhCMV evaded immunity in macaques, a HCMV vaccine must elicit immunity superior to, or different from, that elicited by the prototype RhCMV vaccine to block horizontal transmission.
We report on the implementation of a fully aseptic, single‐use ReadyToProcess™ cross flow filtration system for scalable clarification and concentration of a cytomegalovirus‐like particle (CMV reVLP®), in preclinical development at Redbiotec AG, using ultrafiltration/diafiltration (UF/DF). Performed under gentle feed and permeate flow conditions, with a transmembrane pressure below 0.8 bar and a flux below 30 L m−2 h−1, this trial resulted in a close to 100% product recovery in an overall process time of 3.5 h. During clarification, cell viability was maintained throughout the concentration step (10x), while decreasing marginally during diafiltration (with three diafiltration volumes). The UF/DF process yielded an approximately 13‐fold concentrated material, with high CMV reVLP (virus‐like particle) recovery as determined by the amount of CMV envelope surface protein. We conclude that this system, based on hollow fibers, is a suitable solution for scalable processing of CMV reVLPs, instrumental for a cost‐effective manufacturing process of a future vaccine product. Kept in a fully contained circuit throughout the process, this presterilized system enabled an endotoxin‐free environment by preventing microorganism contamination; moreover, by enabling a fully aseptic process, sterile filtration may possibly be omitted, which can be challenging because of the large size of the CMV reVLPs.
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