Recombinant HCV genotype 1a NS5 protein [GST]

AIM: To investigate whether transfection of plasmid DNA encoding these cytokines enhances both humoral and cellular immune responses to hepatitis C virus (HCV) in a murine model. METHODS: We established a tumor model of HCV infection using syngenic mouse myeloma cells stably transfected with NS5. Co-vaccination of DNA encoding granulocyte macrophage colony-stimulating factor (GMCSF) and Flt-3 ligand together with a plasmid encoding for the HCV NS5 protein was carried out. Mice were sacrificed 14 d after the last immunization event with collection of spleen cells and serum to determine humoral and cellular immune responses. RESULTS: Co-vaccination of DNA encoding GM-CSF and Flt-3 ligand together with a plasmid encoding for the HCV NS5 protein induced increased antibody responses and CD4+ T cell proliferation to this protein. Vaccination with DNA encoding GM-CSF and Flt-3L promoted protection against tumor formation and/or reduction in mice coimmunized with cytokine-encoding DNA constructs. This suggests this strategy is capable of generating cytotoxic T lymphocyte activity in vivo. Following inoculation with plasmid DNA encoding Flt-3L, no increase in spleen size or in dendritic cell (DC) and natural killer cell numbers was observed. This was in contrast to a dramatic increase of both cell types after administration of recombinant Flt3-L in vivo. This suggests that vaccination with plasmid DNA encoding cytokines that regulate DC generation and mobilization may not promote unwanted side effects, such as autoimmunity, splenic fibrosis or hematopoietic malignancies that may occur with administration of recombinant forms of these proteins. CONCLUSION: Our data support the view that plasmid DNA vaccination is a promising approach for HCV immunization, and may provide a general adjuvant vaccination strategy against malignancies and other pathogens. (C) 2006 The WJG Press. All rights reserved.

Infection by hepatitis C virus (HCV)*, the aetiologic agent responsible for the majority of non-A-non-B posttransfusion hepatitis, is detected by assaying for antibodies against structural and nonstructural recombinant proteins or synthetic peptides. The aim of this study was to characterize the antibody reactivity of selected sera against antigenic peptides spanning immunodominant regions of the core, NS4 and NS5 HCV proteins. Reactivity to synthetic peptides was determined by enzyme immunoassay (EIA) for 11 selected sera from blood donors (good responders), for 27 selected sera from hemodialysis patients (poor responders), all positive for HCV antibodies (tested by different second and third-generation assays), and for 7 negative sera. Some peptides from the core and the NS4 region were widely recognized by the tested sera. Sera not reactive with core, NS4, or NS5 region by some immunoblot assays exhibited reactivity against peptides from these proteins. Autoimmune reactivity associated with HCV infection was evaluated by using a synthetic peptide derived from the GOR peptide; 8/11 HCV-positive sera were found reactive against this peptide. No correlation was found between reactivity to any of the peptides tested and the presence of HCV RNA in the serum or with HCV genotype. The EIA reactivity of peptides from the core region suggested a multideterminant antigenic structure, where reactivity of each epitope may be differentially affected by neighboring amino acids depending on individual sera. This situation was particularly evidenced in selected sera from poor responder specimens where a more restricted antibody response to core peptides was observed. Reactivity of sera from HCV-infected patients with synthetic peptides from the core, NS4, and NS5 regions indicated the presence of multiple linear epitopes (particularly in the core region) that may be used in a mixture for immunodiagnosis; however, the length and exact position of the synthetic peptides must be chosen carefully.