title: Low dose and gene gun immunization with a hepatitis C virus nonstructural (NS) 3 DNA-based vaccine containing NS4A inhibit NS3/4A-expressing tumors in vivo.
abstract: The hepatitis C virus (HCV) protease and helicase encompasses the nonstructural (NS) 3 protein and the cofactor NS4A, which targets the NS3/4A-complex to intracellular membranes. We here evaluate the importance of NS4A in NS3-based genetic immunogens. A full-length genotype 1 NS3/4A gene was cloned into a eucaryotic expression vector in the form of NS3/4A and NS3 alone. Transient transfections revealed that the inclusion of NS4A increased the expression levels of NS3. Subsequently, immunization with the NS3/4A gene primed 10- to 100-fold higher levels of NS3-specific antibodies as compared to immunization with the NS3 gene. Humoral responses primed by the NS3/4A gene had a higher IgG2a/IgG1 ratio (>20) as compared to the NS3 gene (3.0), suggesting a T helper 1-skewed response. Low dose i.m. (10 microg) immunization with the NS3/4A gene inhibited the growth of NS3/4A-expressing tumor cells in vivo, whereas the NS3 gene alone or NS3 protein did not. We then evaluated the efficiency of the NS3/4A gene administered by the gene gun, at the same doses used for humans, in priming cytotoxic T lymphocyte (CTL) responses. Three to four 4 microg doses of the NS3/4A gene primed CTL at a precursor frequency of 2-4%, which inhibited the growth of NS3/4A-expressing tumor cells in vivo. Thus, NS4A enhances the expression levels and immunogenicity of NS3, and an NS3/4A gene delivered transdermally could be a therapeutic vaccine candidate.
authors.affiliation: Karolinska Institutet at Huddinge University Hospital, Stockholm, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Frelin
authors.forename: M
authors.initials: M
authors.lastname: Alheim
authors.forename: A
authors.initials: A
authors.lastname: Chen
authors.forename: J
authors.initials: J
authors.lastname: Söderholm
authors.forename: B
authors.initials: B
authors.lastname: Rozell
authors.forename: C
authors.initials: C
authors.lastname: Barnfield
authors.forename: P
authors.initials: P
authors.lastname: Liljeström
authors.forename: M
authors.initials: M
authors.lastname: Sällberg
mesh_terms: D000818:Animals; D019470:Biolistics; D015316:Genetic Therapy / Q000379:methods*; D016174:Hepacivirus / Q000235:genetics*; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009101:Multiple Myeloma / Q000276:immunology / Q000628:therapy* / Q000821:virology; D018709:Statistics, Nonparametric; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D018417:Th1 Cells / Q000276:immunology; D019444:Vaccines, DNA / Q000008:administration & dosage*; D017361:Viral Nonstructural Proteins / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D019444:Vaccines, DNA; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus