Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species

Abstract

Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.

FIG. 1.

Biological assay to quantitate production of infectious adenovirus. Under the same conditions, subconfluent monolayers of primary cultures from the indicated species and tissues were incubated for 1 h with serial dilutions of a replication-competent adenovirus encoding GFP and Ad5 mixed in a VP ratio of 80:1. An inoculation dose of 2 × 10¹¹ VPs/flask translates to an MOI of 200 per cell. After cell monolayers were extensively washed, an aliquot of the supernatants was taken 70 min and 48 h postinfection (pi). As a measurement of infectious virus release, 293 cells were incubated for 24 h with these supernatants, and GFP expression was analyzed by flow cytometry. The percentages of GFP-positive 293 cells 70 min and 48 h postinfection are presented as the means ± standard deviations of at least three independent experiments. Human A549 cells served as controls.

FIG. 2.

Comparative Ad5 transduction efficiency. Indicated cell monolayers were incubated with the same viral inoculum and under the same conditions as described in the legend of Fig. 1. GFP expression was analyzed 24 h later by flow cytometry, and data are presented as the means ± standard deviations of three independent experiments.

FIG. 3.

Adenoviral CPE. Subconfluent monolayers of porcine PK15 cells and primary cultures of porcine kidney cells were infected at an MOI of 5 with Ad5 or mock-infected (control). Seventy-two hours later, cells were photographed through an inverted phase-contrast microscope (original magnification, ×20).

FIG. 4.

Quantitation of adenovirus progeny production in porcine PK15 and human A549 cells by QPCR. Cell monolayers were infected at an MOI of 5 with an E1-deleted Ad.TK vector, an E1B-55K-deleted adenovirus Ad.TK^RC(II) vector, and an E1⁺ but partially E1A-CR2-deleted vector Ad.OW34Δ24. An E1⁺ adenoviral vector Ad.OW34 served as a control. After cell monolayers were extensively washed, the quantity of virus recovered from the medium was assayed 4 and 48 h after infection by QPCR. The increase in adenovirus copy numbers is shown.

FIG. 5.

Biodistribution of adenovirus in swine and mice. Animals received intravenously wild-type Ad5 or a first-generation replication-deficient adenovirus (Ad.TK) at day 0. At 1, 4, and 7 days after virus administration, viral DNA copy numbers in the indicated organs were determined independently in triplicate (swine) or in groups of four mice using QPCR (A). Data are presented as means ± standard deviations. In panel B, the median adenoviral DNA levels determined in triplicate in the blood of individual swine or groups of four mice are shown.

FIG. 6.

Ad5 L2 expression in swine. Seven days after intravenous Ad5 injection, viral hexon expression was analyzed by RT-PCR in lung and liver tissue. mRNA from noninfected PK15 cells and A549 cells infected with Ad5 served as controls. No hexon transcripts were detected in the absence of reverse transcription.

FIG. 7.

Histology. Liver and lungs of swine and mice were histologically evaluated seven days after intravenous Ad5 or Ad.TK injection. Representative micrographs are shown (hematoxylin and eosin staining; original magnification, ×200 (liver) or ×100 (lung).

FIG. 8.

Levels of GLDH in serum in swine. Blood was collected at the indicated time points from swine that received Ad5 or Ad.TK intravenously at day 0. Of the markers indicative of hepatic injury, only GLDH levels changed significantly over the course of the study. The shaded area indicates the reference range of GLDH in swine.