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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

Concomitant of GFP Expression and Cytopathic Effect at Different Times in Infectivity Test(A-D) Infection of 293 permissive cells by Ad-pNS3, expression of GFP and CPE appearance. Thereafter, (A and B) 24 hours after infection of 293 cells and appearance of rounded semi-adherent cells in slide A that appeared as sharp green by fluorescent microscopy in slide B, (C) Overlay image of day two after infection of 293 cells and increased amount of roundedgreen cells on the polygonal cells in  color, (D) Day three-four post infection and devised cells caused a big empty space. Nearly all the cells had signs of GFP expression inside, (E) Hep-G2 cells after infection by AdenopNS3pCore. After two days past infection at MOI 15, fluorescent microscopy showed bright green in more than 70 percent of the cells as a sign of GFP expression, virus infectivity and possibly expression of gene of interest, pNSpCore fused fragment
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fig41: Concomitant of GFP Expression and Cytopathic Effect at Different Times in Infectivity Test(A-D) Infection of 293 permissive cells by Ad-pNS3, expression of GFP and CPE appearance. Thereafter, (A and B) 24 hours after infection of 293 cells and appearance of rounded semi-adherent cells in slide A that appeared as sharp green by fluorescent microscopy in slide B, (C) Overlay image of day two after infection of 293 cells and increased amount of roundedgreen cells on the polygonal cells in color, (D) Day three-four post infection and devised cells caused a big empty space. Nearly all the cells had signs of GFP expression inside, (E) Hep-G2 cells after infection by AdenopNS3pCore. After two days past infection at MOI 15, fluorescent microscopy showed bright green in more than 70 percent of the cells as a sign of GFP expression, virus infectivity and possibly expression of gene of interest, pNSpCore fused fragment

Mentions: Infection of HEK-293 or Hep-G2 cell line with prepared adenoviral vectors showed expression of GFP protein under fluorescent microscopy after 48 hours past infection (Figure 6). To confirm fluorescent microscopy results in detail, Flow cytometery analysis also showed expression of GFP in more than 80% of live population in assessed samples. After infection of HEK-293 cells, adenovirus cytopathic effect (CPE) started to appear 24 hours post infection. As time passed, rounded and swelled cells began to detach from the plate and float in supernatant. After that, on days two-four the CPE grew more by increasing the number of rounded cells, which then fully detached and resulted in creating empty spaces in culture dishes as shown in Figure 6.


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)

Concomitant of GFP Expression and Cytopathic Effect at Different Times in Infectivity Test(A-D) Infection of 293 permissive cells by Ad-pNS3, expression of GFP and CPE appearance. Thereafter, (A and B) 24 hours after infection of 293 cells and appearance of rounded semi-adherent cells in slide A that appeared as sharp green by fluorescent microscopy in slide B, (C) Overlay image of day two after infection of 293 cells and increased amount of roundedgreen cells on the polygonal cells in  color, (D) Day three-four post infection and devised cells caused a big empty space. Nearly all the cells had signs of GFP expression inside, (E) Hep-G2 cells after infection by AdenopNS3pCore. After two days past infection at MOI 15, fluorescent microscopy showed bright green in more than 70 percent of the cells as a sign of GFP expression, virus infectivity and possibly expression of gene of interest, pNSpCore fused fragment
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig41: Concomitant of GFP Expression and Cytopathic Effect at Different Times in Infectivity Test(A-D) Infection of 293 permissive cells by Ad-pNS3, expression of GFP and CPE appearance. Thereafter, (A and B) 24 hours after infection of 293 cells and appearance of rounded semi-adherent cells in slide A that appeared as sharp green by fluorescent microscopy in slide B, (C) Overlay image of day two after infection of 293 cells and increased amount of roundedgreen cells on the polygonal cells in color, (D) Day three-four post infection and devised cells caused a big empty space. Nearly all the cells had signs of GFP expression inside, (E) Hep-G2 cells after infection by AdenopNS3pCore. After two days past infection at MOI 15, fluorescent microscopy showed bright green in more than 70 percent of the cells as a sign of GFP expression, virus infectivity and possibly expression of gene of interest, pNSpCore fused fragment
Mentions: Infection of HEK-293 or Hep-G2 cell line with prepared adenoviral vectors showed expression of GFP protein under fluorescent microscopy after 48 hours past infection (Figure 6). To confirm fluorescent microscopy results in detail, Flow cytometery analysis also showed expression of GFP in more than 80% of live population in assessed samples. After infection of HEK-293 cells, adenovirus cytopathic effect (CPE) started to appear 24 hours post infection. As time passed, rounded and swelled cells began to detach from the plate and float in supernatant. After that, on days two-four the CPE grew more by increasing the number of rounded cells, which then fully detached and resulted in creating empty spaces in culture dishes as shown in Figure 6.

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