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Construction and preparation of three recombinant adenoviruses expressing truncated NS3 and core genes of hepatitis C virus for vaccine purposes.

Hosseini SY, Sabahi F, Moazzeni SM, Modarressi MH, Saberi Firoozi M, Ravanshad M - Hepat Mon (2012)

Bottom Line: The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Tarbiat Modares University, Tehran, IR Iran ; Gastroentero -Hepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran.

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.

No MeSH data available.


Related in: MedlinePlus

Results of Western Blotting Analysis of pNS3 and pNS3pCore proteins Expression by Ad-pNS3 and Ad-pNS3pCore viruses in 293 Cells(A): Western blot analysis of pNS3 protein in 293 cells. 1- Protein Marker 2- Uninfected 293 cells as control negative, 3- Ad-pNS3 virus expressing pNS3 protein with around 32 kd weight (filled triangle), 4- A mix of Control positive and protein marker together, helicase part of NS3 purified protein connected to a special Tag that had been prepared in (J. Lasarte Lab, CIMA, Spain) with estimated 35 kd weight showed by filled triangle standed on 37 kd standard. Prestained Protein marker Cat. Number: 161-0374 (BioRad cop.)(B): Western blot analysis of pNS3 and pNS3pCore proteins expression in 293 cells by Ad-pNS3 and Ad-pNS3pCore virus. 5 and 6- Ad-pNS3pCore virus expressing pNS3pCore protein with estimated 45 kd weight (triangles). 7 and 8- Ad-pNS3 virus expressing pNS3 protein with around 32 kd weight (triangles). 9- Pre-stained Protein marker Cat. Number: 161-0374 (BioRad cop.)
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fig42: Results of Western Blotting Analysis of pNS3 and pNS3pCore proteins Expression by Ad-pNS3 and Ad-pNS3pCore viruses in 293 Cells(A): Western blot analysis of pNS3 protein in 293 cells. 1- Protein Marker 2- Uninfected 293 cells as control negative, 3- Ad-pNS3 virus expressing pNS3 protein with around 32 kd weight (filled triangle), 4- A mix of Control positive and protein marker together, helicase part of NS3 purified protein connected to a special Tag that had been prepared in (J. Lasarte Lab, CIMA, Spain) with estimated 35 kd weight showed by filled triangle standed on 37 kd standard. Prestained Protein marker Cat. Number: 161-0374 (BioRad cop.)(B): Western blot analysis of pNS3 and pNS3pCore proteins expression in 293 cells by Ad-pNS3 and Ad-pNS3pCore virus. 5 and 6- Ad-pNS3pCore virus expressing pNS3pCore protein with estimated 45 kd weight (triangles). 7 and 8- Ad-pNS3 virus expressing pNS3 protein with around 32 kd weight (triangles). 9- Pre-stained Protein marker Cat. Number: 161-0374 (BioRad cop.)

Mentions: After production of virus stocks, gene expression was analyzed by RT-PCR, Q-PCR and western blot. The RT-PCR results indicated suitable expression of all genes after viral transduction into Hep-G2 or 293 and evaluation of total extracted RNA. Western blot analysis also showed suitable expression at protein level in transduced cells. Sharp protein bands were seen after western blot analysis of both recombinant Ad-pNS3 and Ad-pNS3pCore viruses at expected positions on paper (Figure 7). The putative molecular weight of two new proteins by SDS–PAGE was estimated to be around 32 kDa for pNS3 and approximately 45 kDa for pNS3pCore fragment (410 a.a), according to 70 kDa size for the full length NS3 sequence and also another similar study (20). The Real-time PCR for core gene showed good expression of pCore and pNS3pCore by Adenoviruses in Hep-G2 cells in comparison with Adeno-GFP as negative control. Also, the GFP detection in accordance with the mRNA presence was a suitable clue to express the protein due to navigation of both GFP and gene of interest by one strong CMV promoter.


Construction and preparation of three recombinant adenoviruses expressing truncated NS3 and core genes of hepatitis C virus for vaccine purposes.

Hosseini SY, Sabahi F, Moazzeni SM, Modarressi MH, Saberi Firoozi M, Ravanshad M - Hepat Mon (2012)

Results of Western Blotting Analysis of pNS3 and pNS3pCore proteins Expression by Ad-pNS3 and Ad-pNS3pCore viruses in 293 Cells(A): Western blot analysis of pNS3 protein in 293 cells. 1- Protein Marker 2- Uninfected 293 cells as control negative, 3- Ad-pNS3 virus expressing pNS3 protein with around 32 kd weight (filled triangle), 4- A mix of Control positive and protein marker together, helicase part of NS3 purified protein connected to a special Tag that had been prepared in (J. Lasarte Lab, CIMA, Spain) with estimated 35 kd weight showed by filled triangle standed on 37 kd standard. Prestained Protein marker Cat. Number: 161-0374 (BioRad cop.)(B): Western blot analysis of pNS3 and pNS3pCore proteins expression in 293 cells by Ad-pNS3 and Ad-pNS3pCore virus. 5 and 6- Ad-pNS3pCore virus expressing pNS3pCore protein with estimated 45 kd weight (triangles). 7 and 8- Ad-pNS3 virus expressing pNS3 protein with around 32 kd weight (triangles). 9- Pre-stained Protein marker Cat. Number: 161-0374 (BioRad cop.)
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fig42: Results of Western Blotting Analysis of pNS3 and pNS3pCore proteins Expression by Ad-pNS3 and Ad-pNS3pCore viruses in 293 Cells(A): Western blot analysis of pNS3 protein in 293 cells. 1- Protein Marker 2- Uninfected 293 cells as control negative, 3- Ad-pNS3 virus expressing pNS3 protein with around 32 kd weight (filled triangle), 4- A mix of Control positive and protein marker together, helicase part of NS3 purified protein connected to a special Tag that had been prepared in (J. Lasarte Lab, CIMA, Spain) with estimated 35 kd weight showed by filled triangle standed on 37 kd standard. Prestained Protein marker Cat. Number: 161-0374 (BioRad cop.)(B): Western blot analysis of pNS3 and pNS3pCore proteins expression in 293 cells by Ad-pNS3 and Ad-pNS3pCore virus. 5 and 6- Ad-pNS3pCore virus expressing pNS3pCore protein with estimated 45 kd weight (triangles). 7 and 8- Ad-pNS3 virus expressing pNS3 protein with around 32 kd weight (triangles). 9- Pre-stained Protein marker Cat. Number: 161-0374 (BioRad cop.)
Mentions: After production of virus stocks, gene expression was analyzed by RT-PCR, Q-PCR and western blot. The RT-PCR results indicated suitable expression of all genes after viral transduction into Hep-G2 or 293 and evaluation of total extracted RNA. Western blot analysis also showed suitable expression at protein level in transduced cells. Sharp protein bands were seen after western blot analysis of both recombinant Ad-pNS3 and Ad-pNS3pCore viruses at expected positions on paper (Figure 7). The putative molecular weight of two new proteins by SDS–PAGE was estimated to be around 32 kDa for pNS3 and approximately 45 kDa for pNS3pCore fragment (410 a.a), according to 70 kDa size for the full length NS3 sequence and also another similar study (20). The Real-time PCR for core gene showed good expression of pCore and pNS3pCore by Adenoviruses in Hep-G2 cells in comparison with Adeno-GFP as negative control. Also, the GFP detection in accordance with the mRNA presence was a suitable clue to express the protein due to navigation of both GFP and gene of interest by one strong CMV promoter.

Bottom Line: The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Tarbiat Modares University, Tehran, IR Iran ; Gastroentero -Hepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran.

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.

No MeSH data available.


Related in: MedlinePlus