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Characterization of human adenovirus 35 and derivation of complex vectors.

McVey D, Zuber M, Ettyreddy D, Reiter CD, Brough DE, Nabel GJ, King CR, Gall JG - Virol. J. (2010)

Bottom Line: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined.In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness.This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Research, GenVec, Inc, Gaithersburg, MD 20874, USA.

ABSTRACT

Background: Replication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery.

Results: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined. In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness. The packaging capacity of rAd35 was dependent on pIX and vectors were generated with stable genome sizes of up to 104% of the wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors.

Conclusions: The results reported here allow the generation of larger capacity rAd35 vectors and will guide the derivation of adenovirus vectors from other serotypes.

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Related in: MedlinePlus

Viral DNA synthesis. A549 cells were infected with wt Ad35 at an MOI of 5 focus forming units (FFU) per cell, the viral genome number at each time point was determined by qPCR with primers and probe to pIX coding sequences, standardized to total DNA, and expressed as viral genomes per ng of total DNA. Triplicate infections were performed for each time point; standard deviation error bars shown; hpi = hours post infection.
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Figure 2: Viral DNA synthesis. A549 cells were infected with wt Ad35 at an MOI of 5 focus forming units (FFU) per cell, the viral genome number at each time point was determined by qPCR with primers and probe to pIX coding sequences, standardized to total DNA, and expressed as viral genomes per ng of total DNA. Triplicate infections were performed for each time point; standard deviation error bars shown; hpi = hours post infection.

Mentions: Because adenovirus transcriptional patterns differ dramatically before and after viral genome replication [8], we determined the onset of viral DNA replication by QPCR. A549 cells were infected with wild type Ad35 and the number of copies of viral DNA determined at time points from 1 to 48 hours post-infection (hpi). The amount of viral DNA remained unchanged from 1 to 8 hpi and then increased, demonstrating that the onset of viral DNA replication was between 8 and 9 hpi (Figure 2). This information provided the basis to look at differences in viral gene expression between the early and late phases of viral infection. Taken together, these data allowed for subsequent identification of effects of deletions on both transcription and protein expression levels.


Characterization of human adenovirus 35 and derivation of complex vectors.

McVey D, Zuber M, Ettyreddy D, Reiter CD, Brough DE, Nabel GJ, King CR, Gall JG - Virol. J. (2010)

Viral DNA synthesis. A549 cells were infected with wt Ad35 at an MOI of 5 focus forming units (FFU) per cell, the viral genome number at each time point was determined by qPCR with primers and probe to pIX coding sequences, standardized to total DNA, and expressed as viral genomes per ng of total DNA. Triplicate infections were performed for each time point; standard deviation error bars shown; hpi = hours post infection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984591&req=5

Figure 2: Viral DNA synthesis. A549 cells were infected with wt Ad35 at an MOI of 5 focus forming units (FFU) per cell, the viral genome number at each time point was determined by qPCR with primers and probe to pIX coding sequences, standardized to total DNA, and expressed as viral genomes per ng of total DNA. Triplicate infections were performed for each time point; standard deviation error bars shown; hpi = hours post infection.
Mentions: Because adenovirus transcriptional patterns differ dramatically before and after viral genome replication [8], we determined the onset of viral DNA replication by QPCR. A549 cells were infected with wild type Ad35 and the number of copies of viral DNA determined at time points from 1 to 48 hours post-infection (hpi). The amount of viral DNA remained unchanged from 1 to 8 hpi and then increased, demonstrating that the onset of viral DNA replication was between 8 and 9 hpi (Figure 2). This information provided the basis to look at differences in viral gene expression between the early and late phases of viral infection. Taken together, these data allowed for subsequent identification of effects of deletions on both transcription and protein expression levels.

Bottom Line: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined.In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness.This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Research, GenVec, Inc, Gaithersburg, MD 20874, USA.

ABSTRACT

Background: Replication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery.

Results: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined. In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness. The packaging capacity of rAd35 was dependent on pIX and vectors were generated with stable genome sizes of up to 104% of the wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors.

Conclusions: The results reported here allow the generation of larger capacity rAd35 vectors and will guide the derivation of adenovirus vectors from other serotypes.

Show MeSH
Related in: MedlinePlus