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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

Fiber Amplification as a Confirmatory Step of Recombinant Virus PresenceAfter two step of reinfection, supernatant or lysed cells undergo a 25 cycle PCR reaction to amplify 1700 bp from fiber gene as virus production sign. As shown, no significant difference was seen between the sample processed by standard lyses method or supernatant medium. From left to right, A) sample from supernatant without lyses B) sample prepared by standard lyses method C) 293 negative control.
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fig39: Fiber Amplification as a Confirmatory Step of Recombinant Virus PresenceAfter two step of reinfection, supernatant or lysed cells undergo a 25 cycle PCR reaction to amplify 1700 bp from fiber gene as virus production sign. As shown, no significant difference was seen between the sample processed by standard lyses method or supernatant medium. From left to right, A) sample from supernatant without lyses B) sample prepared by standard lyses method C) 293 negative control.

Mentions: In order to screen the formation and amplification of first viral particles, cultured cells were evaluated to detect GFP emission every two days. During the first three-four days after transfection of linear Adenovirus constructs into cells, there was a GFP pattern such as the one observed in traditional transfection (Figure 3). After five-six days, as a consequence of spreading the released viruses through neighboring cells in culture dishes the pattern of foci appeared as a clue for reproductive amplification of recombinant viral particles as shown in Figure 3. Special view of comet-like fluorescence or a focus forming pattern under fluorescent microscopy at days five-seven post transfection served as an important clue for vector production as explained before (37).To evaluate the presence of the viral genome as a direct sign of virus production, PCR test for fiber of adenoviral vector was developed. The PCR with fiber specific primers on cell lysate showed a sharp 1700 bp fragment, indicating the propagation of Adenovirus particles after two cycles of reinfection with 293 cell line. Interestingly, no significant difference was seen between the PCR results of the samples prepared from the cells by proteinase K lysis method or untreated culture supernatant (Figure 4). Electron microscopy was employed for detailed assessment of the production of viral particles in all virus batches. Electron microscopy revealed many Adenoviruses like particles in clusters or single harboring faint fibers projected from the surroundings of the particles (Figure 5). Among these complete Adenoviruses, many defective particles were also detected with similar shape but smaller size at the background. After viral purification, titer of recombinant viral stocks was evaluated as both particle/ml and Infectious particle/ml as described under the mentioned methods. The results indicated that the ratio of infectious particles to total particle counts was less than 1/200 in all virus batches. The real titers of these batches were calculated to be around 109 infectious units/ml.


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)

Fiber Amplification as a Confirmatory Step of Recombinant Virus PresenceAfter two step of reinfection, supernatant or lysed cells undergo a 25 cycle PCR reaction to amplify 1700 bp from fiber gene as virus production sign. As shown, no significant difference was seen between the sample processed by standard lyses method or supernatant medium. From left to right, A) sample from supernatant without lyses B) sample prepared by standard lyses method C) 293 negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig39: Fiber Amplification as a Confirmatory Step of Recombinant Virus PresenceAfter two step of reinfection, supernatant or lysed cells undergo a 25 cycle PCR reaction to amplify 1700 bp from fiber gene as virus production sign. As shown, no significant difference was seen between the sample processed by standard lyses method or supernatant medium. From left to right, A) sample from supernatant without lyses B) sample prepared by standard lyses method C) 293 negative control.
Mentions: In order to screen the formation and amplification of first viral particles, cultured cells were evaluated to detect GFP emission every two days. During the first three-four days after transfection of linear Adenovirus constructs into cells, there was a GFP pattern such as the one observed in traditional transfection (Figure 3). After five-six days, as a consequence of spreading the released viruses through neighboring cells in culture dishes the pattern of foci appeared as a clue for reproductive amplification of recombinant viral particles as shown in Figure 3. Special view of comet-like fluorescence or a focus forming pattern under fluorescent microscopy at days five-seven post transfection served as an important clue for vector production as explained before (37).To evaluate the presence of the viral genome as a direct sign of virus production, PCR test for fiber of adenoviral vector was developed. The PCR with fiber specific primers on cell lysate showed a sharp 1700 bp fragment, indicating the propagation of Adenovirus particles after two cycles of reinfection with 293 cell line. Interestingly, no significant difference was seen between the PCR results of the samples prepared from the cells by proteinase K lysis method or untreated culture supernatant (Figure 4). Electron microscopy was employed for detailed assessment of the production of viral particles in all virus batches. Electron microscopy revealed many Adenoviruses like particles in clusters or single harboring faint fibers projected from the surroundings of the particles (Figure 5). Among these complete Adenoviruses, many defective particles were also detected with similar shape but smaller size at the background. After viral purification, titer of recombinant viral stocks was evaluated as both particle/ml and Infectious particle/ml as described under the mentioned methods. The results indicated that the ratio of infectious particles to total particle counts was less than 1/200 in all virus batches. The real titers of these batches were calculated to be around 109 infectious units/ml.

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