Construction and Preparation of Three Recombinant Adenoviruses Expressing Truncated NS3 and Core Genes of Hepatitis C Virus for Vaccine Purposes

authors:

avatar Seyed Younes Hosseini 2 , avatar Farzaneh Sabahi 1 , * , avatar Seyed Mohammad Moazzeni 3 , avatar Mohammad Hossein Modarressi 4 , avatar Mehdi Saberi Firoozi 5 , avatar Mehrdad Ravanshad 1

Gastroentero -Hepatology Research Center, Shiraz University of Medical Sciences, IR Iran
Department of Virology, Tarbiat Modares University, sabahi_f@modares.ac.ir, IR Iran
Department of Immunology, Tarbiat Modares University, IR Iran
Department of Medical Genetic, Tehran University of Medical Sciences, IR Iran
Digestive Disease Research Center, Tehran University of Medical Sciences, IR Iran
Hepatitis Monthly: Vol.12, issue 8; 6130
published online: August 14, 2012
article type: Research Article
received: May 16, 2012
accepted: June 26, 2012
DOI: https://doi.org/10.5812/hepatmon.6130

how to cite: Hosseini S, Sabahi F, Moazzeni S, Modarressi M, Saberi Firoozi M, et al. Construction and Preparation of Three Recombinant Adenoviruses Expressing Truncated NS3 and Core Genes of Hepatitis C Virus for Vaccine Purposes. Hepat Mon. 2012;12(8):6130. https://doi.org/10.5812/hepatmon.6130.

Abstract

Background:

In spite of dozens of clinical trials to establish effective therapeutic and/or preventive vaccine to resolve HCV infection, no real vaccine has been proved to date. Genetic vaccines based on replication-defective adenoviruses have proved to elicit strong and long lasting T-cell responses against a number of viral antigens and are even currently being used for vaccine trials in humans. According to the controversy in the immune modulatory effects of both core and NS3 full length genes, it seemed more practical to employ some parts of these HCV proteins for vaccine design.

Objectives:

To generate recombinant Adenoviral vectors containing new overlapping-truncated region of NS3 gene or both the N- and C-terminal deleted parts of core gene, as well as a fusion fragment derived from both of them.

Materials and Methods:

The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared. The integrity and sequence of the transfer vectors were confirmed, and followed by experiments involving homologous recombination between them and the adenovirus backbone plasmid in the bacterial host. Recombinant Ad-pNS3, Ad-pCore and Ad-pNS3pCore viruses were prepared by transfection of these new recombined constructs into 293 packaging cell lines. The virus titer was then calculated by an immunohistochemistry based method. The RT-PCR, Real-Time PCR and western blotting were used to evaluate gene expression by all recombinant constructs. The production of complete virion particles was evaluated by detailed electron microscopy in addition to the appearance of typical cytopathic effects (CPE) and GFP expression patterns in 293 cells. The RT-PCR and GFP detection were employed to monitor the integrity as well as infectivity potency of the viral particles in Hep-G2 cells.

Results:

RT-PCR, Real-Time PCR or western blotting confirmed expression of truncated fragment of NS3, core or a fusion fragment of theirs by newly constructed Ad-pNS3, Ad-pCore, Ad- pNS3pCore particles. Electron microscopy, which revealed many adenovirus-like particles and characteristics of CPE in infected cells in addition to GFP detection, confirmed the infectivity, potency and integrity of recombinant adenoviral particles.

Conclusions:

These adenoviruses expressing novel fragments of NS3 and core genes may be suitable tools to overcome shortcomings associated with full gene expression in the setting of HCV vaccine therapy.

Full Text

Full text is available in PDF