Limits...
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

Schematic Representation of Homologous Recombination Between Constructed Shuttle Vector (IR-pNS3-pCore) and pAdenoVator in BJ Bacterial HostLinearized shuttle plasmid together with supercoiled pAdenovator co-transformed into bacterial host to lead recombination. Finally, the created recombinant Ad-pNS3-pCore vector contains all shuttle plasmid elements rather than adenovirus backbone. Recombination lead to replacement of all auxiliary genetic elements inside the pAdenoVator with those of linearized shuttle plasmid as described in the text.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3475015&req=5

fig36: Schematic Representation of Homologous Recombination Between Constructed Shuttle Vector (IR-pNS3-pCore) and pAdenoVator in BJ Bacterial HostLinearized shuttle plasmid together with supercoiled pAdenovator co-transformed into bacterial host to lead recombination. Finally, the created recombinant Ad-pNS3-pCore vector contains all shuttle plasmid elements rather than adenovirus backbone. Recombination lead to replacement of all auxiliary genetic elements inside the pAdenoVator with those of linearized shuttle plasmid as described in the text.

Mentions: Amplification of new partial core (aa 50-160), new overlapping region of NS3 gene covering protease/helicase domains and a fusion of both truncated fragments from HCV genotype 1a were previously described (30, 31). In brief, full core and partial NS3 sequences were propagated from the serum of a HCV patient by RT-PCR. The resulting extracted amplicons were first cloned into the pTZ57R/T cloning vector. After primary confirmation of the prepared core gene, to amplify partial core harboring amino acid sequence 50-160 which is an N and C-terminal deleted sequence, a pair of primers containing Nde-I restriction site was exploited on a pTZ57R/T vector containing full core gene. The resultant amplicon of core gene was inserted into the TA-pNS3 construct at NdeI site, making a new fusion sequence of pNS3 and pCore. Finally, the gene fragments were inserted into pAdenovator-CMV5-IRES-EGFP vector as shuttle vector. The transfer/shuttle vectors contained partial NS3 or partial core and fusion of both genes designated as IR-pNS3, IRpCore, and IR-pNS3-pCore (Figure 1) respectively. Phylogenetic analysis of sequences was also used to confirm true cloning of the desired genotype as described previously (30, 31).


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)

Schematic Representation of Homologous Recombination Between Constructed Shuttle Vector (IR-pNS3-pCore) and pAdenoVator in BJ Bacterial HostLinearized shuttle plasmid together with supercoiled pAdenovator co-transformed into bacterial host to lead recombination. Finally, the created recombinant Ad-pNS3-pCore vector contains all shuttle plasmid elements rather than adenovirus backbone. Recombination lead to replacement of all auxiliary genetic elements inside the pAdenoVator with those of linearized shuttle plasmid as described in the text.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475015&req=5

fig36: Schematic Representation of Homologous Recombination Between Constructed Shuttle Vector (IR-pNS3-pCore) and pAdenoVator in BJ Bacterial HostLinearized shuttle plasmid together with supercoiled pAdenovator co-transformed into bacterial host to lead recombination. Finally, the created recombinant Ad-pNS3-pCore vector contains all shuttle plasmid elements rather than adenovirus backbone. Recombination lead to replacement of all auxiliary genetic elements inside the pAdenoVator with those of linearized shuttle plasmid as described in the text.
Mentions: Amplification of new partial core (aa 50-160), new overlapping region of NS3 gene covering protease/helicase domains and a fusion of both truncated fragments from HCV genotype 1a were previously described (30, 31). In brief, full core and partial NS3 sequences were propagated from the serum of a HCV patient by RT-PCR. The resulting extracted amplicons were first cloned into the pTZ57R/T cloning vector. After primary confirmation of the prepared core gene, to amplify partial core harboring amino acid sequence 50-160 which is an N and C-terminal deleted sequence, a pair of primers containing Nde-I restriction site was exploited on a pTZ57R/T vector containing full core gene. The resultant amplicon of core gene was inserted into the TA-pNS3 construct at NdeI site, making a new fusion sequence of pNS3 and pCore. Finally, the gene fragments were inserted into pAdenovator-CMV5-IRES-EGFP vector as shuttle vector. The transfer/shuttle vectors contained partial NS3 or partial core and fusion of both genes designated as IR-pNS3, IRpCore, and IR-pNS3-pCore (Figure 1) respectively. Phylogenetic analysis of sequences was also used to confirm true cloning of the desired genotype as described previously (30, 31).

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