title: Construction and Immunogenicity Analysis of Hepatitis C Virus (HCV) Truncated Non-Structural Protein 3 (NS3) Plasmid Vaccine.
abstract: BACKGROUND
  To develop hepatitis C virus (HCV) vaccine, induction of potent humoral and T cell response against immunogenic targets with conserved region should be achieved. T cell response against NS3 is often associated with complete clearance of the virus.
  
  
  OBJECTIVE
  Herein, we expressed the truncated form of NS3 in a mammalian cell line and evaluated immune responses of NS3 DNA vaccine in BALB/c.
  
  
  METHODS
  The partial length of NS3 gene, which encodes immunogenic epitopes (1095 - 1379 aa), was amplified by reverse transcription-polymerase chain reaction (RT-PCR) on RNA obtained from a patient with HCV, inserted into pcDNA3.1 plasmid using XhoI/HindIII sites, and finally evaluated by restriction analysis and sequencing. After transfection of the recombinant plasmid into HEK293T cells, the NS3 protein expression was confirmed by western blotting. Mice were immunized intra-dermally close to the base of the mice tail with four doses in two-weeks intervals and the immune responses were assessed using total and subtypes of IgG antibody assay, cell proliferation and cytokine assay.
  
  
  RESULTS
  The pcDNA3.1 plasmid harboring the coding sequence of NS3 (pc-NS3) was constructed and confirmed with the expected size. Proper expression of the recombinant protein in transfected HEK 293T cells was confirmed using western blotting. The immunization results indicated that pc-NS3 induced significant levels of total antibody, IgG2a subclass antibody, Interferon (IFN)-γ, Interleukin (IL)-4 and proliferation assay compared to the control group (P < 0.05).
  
  
  CONCLUSIONS
  The pc-NS3 possesses the capacity to express NS3 in the mammalian cell line and demonstrated strong immunogenicity in a murine model. Our primary results demonstrated that the immunogenic truncated region of NS3 could be used as a potential vaccine candidate against hepatitis C.
authors.affiliation: Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
authors.forename: Mohammad-Hassan
authors.initials: MH
authors.lastname: Pouriayevali
authors.affiliation: Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
authors.forename: Taravat
authors.initials: T
authors.lastname: Bamdad
authors.affiliation: Department of Hepatitis and AIDS, Pasteur Institute of Iran. Tehran, IR Iran.
authors.forename: Mohammad-Reza
authors.initials: MR
authors.lastname: Aghasadeghi
authors.affiliation: Department of Hepatitis and AIDS, Pasteur Institute of Iran. Tehran, IR Iran.
authors.forename: Seyed Mehdi
authors.initials: SM
authors.lastname: Sadat
authors.affiliation: Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
authors.forename: Farzaneh
authors.initials: F
authors.lastname: Sabahi
keywords: DNA Vaccine; Hepatitis C Virus; Immunization; NS3; Plasmid