Genetic Immunization of Wild-Type and Hepatitis C Virus Transgenic Mice Reveals a Hierarchy of Cellular Immune Response and Tolerance Induction against Hepatitis C Virus Structural Proteins

Abstract

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...

Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection

“…P7020.S expressing the core͞E1͞E2 polyprotein (amino acids 1-830) under the control of the cytomegalovirus promoter was constructed by cloning the same HCV cDNA from the bvHCV.S as described (18). Plasmid DNA was purified by using an endotoxin-free plasmid extraction kit (Qiagen, Valencia, CA) and dissolved in PBS at a concentration of 2 mg͞ml.…”

“…Responder cells (3 ϫ 10 7 ) in 5 ml of CTM with 10% rat T-stim were cocultured with 10 g͞ml HCV core (amino acids 131-140, ADLMGYIPLV) or E2 (amino acids 614-622, RLWHYPCTI) peptide (18) in a six-well plate in a humidified incubator at 37°C, 5% CO 2 . Five days after stimulation, cytolytic activity of in vitrostimulated CTLs was measured by using a 6-h assay with 51 Crlabeled target cells.…”

Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene

“…It has been shown that DNA immunizations can induce both specific antibodies and cell-mediated responses against the structural and nonstructural (NS) HCV proteins in mice. [6][7][8][9][10][11][12][13][14][15][16] The NS protein 3 of HCV has been considered as a possible vaccine target. The reasons for this are that the NS3 protein shows a limited genetic variability, it performs multiple enzymatic functions, and it is a relatively large protein.…”

Prophylactic and therapeutic vaccination with dendritic cells against hepatitis C virus infection

“…Peptides were proposed by computer simulation (University of Wisconsin Genetics Computer Group (UWGCG), peptide structure program) and prepared by EMC microcollections (Tübingen, Germany) with the following sequences: YQVRNSSGLYHVTNDCPNSS (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), PGCVPCVREGNAS RCWVAVT (33-53), REGNASRCWVAVTPTVATRDGKL and PRRHWTTQDCNCSIYPG (104-120). Establishment and characterization of the stable transfected cell line expressing HCV core protein (SP2-19) and the control cell line SP2-0 have been described previously [34].…”

“…induction of only borderline immunity after DNAimmunization or safety issues with viral vectors [15,16]. A number of different vaccine approaches have been investigated against HCV infection in the past like DNA vaccination [17][18][19], vaccination with recombinant proteins or peptides [11], vaccines based on virus-like particles [20] and viral or other pathogen vectors containing genetic information of HCV proteins [21].…”