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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 Hepatitis C virus (HCV) is a positive-stranded RNA virus that belongs to the Flaviviridae family. The HCV genome contains a single open reading frame coding for a polyprotein that is cleaved into structural and nonstructural proteins by hostand virus-specific proteases. The structural proteins consist of core and 2 envelope glycoproteins E1 and E2. Given the global health burden of HCV infection, there is a paramount need to develop a vaccine against HCV. 1 However, a robust infectious tissue culture system for passaging and expanding the virus and for testing neutralizing antibodies is still lacking. Therefore, newer approaches have to be adopted for HCV vaccine development.Because HCV is an enveloped virus, neutralizing determinants likely reside on the surface of the envelope. The envelope protein E2 of HCV contains highly variable sequences within the N-terminal region (HVR1), which are thought to contain neutralizing B-cell epitopes. 2 Immunization of chimpanzees with recombinant E1/E2 proteins can induce hightiter anti-envelope antibodies but only protects animals from a low-dose viral challenge with the homologous strain. 3 Studies in humans and chimpanzees have indicated that failure to generate multispecific cellular immune responses against HCV in the acute phase of infection is associated with chronicity. 4-7 Therefore, an ideal HCV vaccine should be able to induce strong humoral immune responses against the envelope proteins and to prime broad, HCV-specific T-helper and cytotoxic T-cell responses (for review, see Houghton 8 and Lechmann and Liang 9 ).Virus-like particles are attractive as a recombinant protein vaccine, because they mimic closely the properties of native virions. Papillomavirus-and rotavirus-like particles synthesized in insect cells have been shown to generate protective immunity. [10][11][12] These studies showed that virus-like particles can induce not only high-titer neutralizing antibodies but also strong cytotoxic T lymphocyte (CTL) responses in immunized animals. [13][14][15] Our laboratory has recently reported the synthesis of hepatitis C virus-like particles in insect cel...

Serum TPO level may not be directly associated with thrombocytopenia in patients with chronic hepatitis and liver cirrhosis. In contrast, spleen volume and PAIgG are associated with thrombocytopenia in such patients, suggesting that hypersplenism and immune-mediated processes are predominant thrombocytopenic mechanisms.

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

To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.Transgenic models have been developed to study the mechanisms of tolerance and their implications for autoimmune or other immune-mediated diseases. In this regard, transgeneencoded neo-self antigen coupled with the corresponding Tcell receptor transgene has been particularly valuable (4,20,23,35). In addition to central thymic selection, peripheral tolerance mechanisms, including peripheral deletion, anergy, and ignorance have been defined (5,10,27,28,36). In the latter case, it is often possible to break tolerance and induce autoimmunity, leading to immune-mediated tissue injury. Expression of neo-self antigens in the liver presents a particular interesting scenario because of the putative toleragenic role of the liver in immune response and the unique anatomy of the liver in which the fenestrated vasculature allows direct access of hepatocytes to circulating T cells (25). This intriguing question has been addressed in several transgenic models in which central and peripheral deletion of reactive T cells appears to confer a robust tolerance to the neo-self antigen expressed in the transgenic liver. In situations whereby peripheral anergy or ignorance induction is operative, tolerance at the T-cell level can be broken by either viral infection or dendritic cell or DNA immunization (23,31,35,37). However, induction of hep...

Adaptive epigenetic changes and toxicity often accompany constitutive expression of a transgene or knockout of an endogenous gene in mice. These considerations potentially limit the usefulness of transgenic technology in studying the in vivo functions of a gene. Using conditional gene expression technology, it is possible to override such restrictions to achieve temporal and tissue-specific manipulation of gene expression in vivo. Based on the tetracycline regulatory system, we established a binary transgenic model in which the conditional expression of two transgenes, SV40 T antigen (TAg) and lacZ, can be tightly regulated in the liver by administration of tetracycline. The mouse albumin or mouse major urinary protein promoter was used to achieve liver-specific expression of the tetracycline-responsive transcriptional activator (tTA) in one set of transgenic mice. These mice were crossed with transgenic mice carrying either TAg or lacZ under the control of the tTA-regulated promoter. Analyses of mice transgenic for both tTA and TAg (or lacZ) revealed that the liver-specific expression of the transgenes could be suppressed to undetectable levels and regulated in a reversible fashion by tetracycline administration and withdrawal. Mice with tTA and TAg transgenes developed hepatocellular adenomas and hyperplasia that could be prevented by continuous tetracycline administration. Our report demonstrates the value of this binary transgenic model in studying the physiological functions of any potential genes of interest in a liverspecific manner.

We evaluated the effect of a ''tailor-made'' chemo-gene therapy in scirrhous gastric cancer (SGC)-bearing nude mice. For this tailormade approach, we first selected gefitinib (epidermal growth factor receptor-tyrosine kinase inhibitor)-sensitive SGC cell lines, and 5/8 cell lines demonstrated various degrees of gefitinib-sensitivity. In the highly gefitinib-sensitive NUGC-4, the biological response to NK4 (HGF antagonist/angiogenesis inhibitor) was examined. Subsequently, the composition of an NK4-expressing ternary complex (cationic lipid/nucleic acid/HMG-1, 2 protein) was optimized for maximum transfection activity in NUGC-4. Finally, mice were peritoneally coinoculated with NUGC-4 and scirrhous-associated gastric fibroblasts, NF22, on day 0. Animal models were orally administrated gefitinib (50 mg/kg/day, on days 7-28), and peritoneally NK4-expressing ternary complex (on days 14, 21 and 28). NK4-expression suppressed the gefitinib-resistance induced by the interaction between fibroblasts and SGC, and eventually, this tailor-made combination synergistically decelerated the disease progression by inhibiting proliferative, angiogenic and antiapoptotic effects in tumor tissues. On day 28, both the hemoglobin concentration (g/dl) (control (n 5 8), 11.9; treated (n 5 8), 17.3; p 5 0.0014) and the numbers of mice in good condition (control, 2; treated, 8; p 5 0.0012) were significantly greater, and the abdominal girth (mm) (control, 81.1; treated, 70.3; p 5 0.0036) was significantly reduced. The median points of bloody ascite-free survival time (days) (control, 22; treated, 44; p < 0.0001) and time to euthanasia (days) (control, 36.5; treated, 56; p < 0.0001) were also significantly prolonged. This combination is a potentially useful approach to the treatment of peritoneal gefitinib-sensitive SGC dissemination. ' 2005 Wiley-Liss, Inc.

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