DNA immunization with a plasmid carrying the gene of hepatitis C virus protein 5A (NS5A) induces an effective cellular immune response

Abstract

In spite of extensive research, no effective vaccine against hepatitis C virus (HCV) has been devel oped so far. DNA immunization is a potent technique of vaccine design strongly promoting the cellular arm of immune response. The genes encoding nonstructural HCV proteins (NS2-NS5B) are promising candi dates for vaccine development. NS5A is a protein involved in viral pathogenesis, in the induction of immune response, and probably in viral resistance to interferon treatment. The objective of this study was to construct a DNA vaccine encoding NS5A protein and evaluate its immunogenicity. A plasmid encoding a full size NS5A protein was produced using the pcDNA3.1 (+) vector for eukaryotic expression system. The expression of the NS5A gene was confirmed by immunoperoxidase staining of the transfected eukaryotic cells with anti NS5A monoclonal antibodies. Triple immunization of mice with the plasmid vaccine induced a pronounced cellular immune response against a broad spectrum of NS5A epitopes as assessed by T cell proliferation and secretion of antiviral cytokines IFN γ and IL 2. In T cell stimulation in vitro experiments, NS5A derived antigens were modeled by synthetic peptides, recombinant proteins of various genotypes, and phages carrying exposed NS5A peptides. A novel immunomodulator Immunomax showed high adjuvant activity in DNA immunization. The data obtained indicate that the suggested DNA construct has a strong potential in the development of the gene vaccines against hepatitis C.

A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

“…Because of the limited knowledge of the immunogenic properties of NS5A, we sought to study its intrinsic properties. The limited number of studies of the intrinsic properties of NS5A as a genetic immunogen has demonstrated poor immunogenicity (17)(18)(19). Moreover, only a limited number of HLA-A2 CD8 + T cell-restricted epitopes have been identified (16,51,52).…”

“…To be able to characterize cellular immune responses to NS5A in H-2 b mice, we first had to identify MHC class I epitopes within NS5A. Several MHC class I epitopes are described in other mouse haplotypes (17)(18)(19), but to our knowledge, none is described for H-2 b . We therefore used overlapping peptides covering the complete NS5A gene combined with epitope predictions.…”

“…The HCV NS5A is of particular interest because it is recognized by cytotoxic T lymphocytes during natural infection and resolution of acute HCV in humans (16). Thus, we decided to evaluate HCV NS5A as a therapeutic DNA vaccine candidate for chronic HCV infection, because relatively few studies have investigated NS5A as a potential vaccine (17)(18)(19). The activation of a potent antiviral T cell response is a prerequisite for a functional therapeutic vaccine in the presence of the homologous virus.…”

The combined application of nucleotide and amino acid sequences of NS3 hepatitis C virus protein, DNA encoding granulocyte macrophage colony-stimulating factor, and inhibitor of regulatory T cells induces effective immune responce against Hepatitis C virus

“…T cell proliferation was used to detect the in vitro T cell response by measur ing levels of IFN γ and IL 2 cytokines, as well as ELISpot assay. Splenic cells from five mice of each group were suspended, and the fraction of mononu clear cells was isolated as described previously [17,18]. An analysis of the cellular reactions was con ducted in triplicate wells.…”

“…Plasmid pcDNA NS3 was tested for its capacity to express a NS3 HCV gene in mammalian cells using cell line derived from African green monkey kidney (Vero) cells. Cells were cultivated as described previously [17]. 5 × 10 4 cells were grown on 24 well plates (Nunc, Denmark) 24 h before transfection.…”