Development of a method for effective amplification of human adenovirus 40

Abstract

Human adenovirus 40 (Ad40) is an interesting candidate for vector construction because of its tropism for the gastrointestinal tract. Although effective preparation of the vector is necessary for its in vivo application, amplification of Ad40 has been very difficult. Ad40 E1 deletion mutants were detected by PCR in the viral DNA from Ad40 Dugan amplified by Ad5 E1-expressing human embryonic kidney (293) cells and in Ad40 Dugan plaques observed with Ad5 E1-expressing human retinoblastic cells. For the purpose of generating a single wild-type Ad40 clone, the entire Ad40 DNA was cloned into a plasmid by homologous recombination. A pure Ad40 was successfully generated by plasmid transfection and subsequently amplified with Ad5 E4orf6-inducible 293 (2V6.11) cells. 2V6.11 is an apposite cell line for effective Ad40 amplification and for future vector construction because Ad40 genetic integrity was maintained with this Ad5 E1 and E4orf6 trans-complementing cell line.

Fig. 1

E1 region of Ad40 Dugan-derived mutants. (a) PCR detection of E1 deletion mutants derived from Ad40 Dugan. The picture shown is representative of more than three independent experiments on the encapsidated viral DNA extracted from Ad40 Dugan obtained from ATCC and from a Ad40 Dugan plaque isolated with 911 cells. The expected DNA size with intact Ad40 E1 is shown on the right of the picture. (b) Schematic representation of the internal tandem repeat (ITR), TATA box, E1A, E1b19K and E1b55K region on the left-hand end of Ad40. The nucleotide number shown was based on GenBank accession no. L19443. (c) Schematic view of Ad40 Dugan-derived mutants. The deleted segments were indicated by dotted lines. The nucleotide number shown was the left- and right-hand breakpoints of deletions (deletion length, bp). * An Ad40 E1A- and E1b55K-deletion mutant in Ad40 Dugan plaques isolated with 911 cells; mRNA, messenger RNA; CDS, coding sequencing.

Fig. 1

E1 region of Ad40 Dugan-derived mutants. (a) PCR detection of E1 deletion mutants derived from Ad40 Dugan. The picture shown is representative of more than three independent experiments on the encapsidated viral DNA extracted from Ad40 Dugan obtained from ATCC and from a Ad40 Dugan plaque isolated with 911 cells. The expected DNA size with intact Ad40 E1 is shown on the right of the picture. (b) Schematic representation of the internal tandem repeat (ITR), TATA box, E1A, E1b19K and E1b55K region on the left-hand end of Ad40. The nucleotide number shown was based on GenBank accession no. L19443. (c) Schematic view of Ad40 Dugan-derived mutants. The deleted segments were indicated by dotted lines. The nucleotide number shown was the left- and right-hand breakpoints of deletions (deletion length, bp). * An Ad40 E1A- and E1b55K-deletion mutant in Ad40 Dugan plaques isolated with 911 cells; mRNA, messenger RNA; CDS, coding sequencing.

Fig. 2

Encapsidated viral DNA levels of pure Ad40wt during propagation. Pure Ad40wt was generated from plasmid transfection and amplified onto 2V6.11 cells with 1 μg/ml ponasterone A. In a 60-mm dish every 72 h until the appearance of obvious CPE (9^th passage), the same amplification was repeated using the 1:3 dilutions of CVLs (1.5 ml into 3 ml fresh medium), which were prepared by centrifugation (1500 rpm, 10 min), reduced the volumes from 4.5 ml to 1.7 ml, three freeze-thawed and centrifuged (3000 rpm, 10 min). After observing CPE and confirming the existence of Ad40 E1, fiber, and E4orf2 by PCR, the virus was amplified continuously for CsCl centrifugation. The Ad40 fiber DNA levels were evaluated by real-time PCR on the encapsidated viral DNA from the 200 μl CVLs at each passage (in triplicate). The results shown are mean values of triplicates. Error bars indicate the 95% confidence interval.

Fig. 3

E1 and fiber DNA of pure Ad40wt. Cells were infected with 200 VP/cell of pure Ad40wt. At 72 h pi, encapsidated viral DNA from the the 200 μl supernatants (SNs) and CVLs was purified and Ad40 E1 DNA was tested by PCR on encapsidated viral DNA from the 200 μl CVLs. The results of three independent experiments are shown on the top of figures (a). Plus (+) means reproducible PCR-positive, minus (−) means reproducible PCR-negative, and plus-minus (±) means unreproducible PCR-positive. The Ad40 fiber DNA levels (copy numbers per ml) in the SNs (0 h pi; on the left hand, empty bar graph and 72 h pi; in the middle, shaded bar graph) and CVLs (72 h pi; on the right, black bar graph) from pure Ad40wt were performed by real-time PCR (a). The results shown are mean values of three independent experiments. Error bars indicate the 95% confidence interval. The picture shown is representative of three independent experiments on pure Ad40wt infected 2V6.11 cells (b). The expected DNA size with intact Ad40 E1 is shown on the right of the picture. U, uninduced control (0.1% DMSO). PonA 1 or 5, 1 or 5 μg/ml ponasterone A.

Fig. 4

Expression of Ad5 E1b55K or Ad5 E4orf6 in 2V6.11 or 911orf6 cells, and Mre11 expression in 2V6.11 cells. Cells were treated with 0.1% DMSO (U, uninduced control), 1 or 5 μg/ml ponasterone A (PonA 1 or PonA 5), or 200 VP/cell Ad5wt. At 24 h pi, cells were harvested (a) or cells were infected with virus-free culture medium, 200 or 2000 VP/cell pure Ad40wt, or 200 VP/cell Ad5wt (b). Cell extracts were analyzed by SDS-PAGE followed by Western blotting using approximate antibodies as indicated to the right. Expected protein sizes are shown on the left of pictures.