1. Volume 14, Issue 1, Pages 118-128 (July 2006)
Adenovirus Type 5 Interactions with Human Blood Cells May Compromise Systemic Delivery 
Mark Lyons, David Onion, Nicky K. Green, Kriss Aslan, Ratna Rajaratnam, Miriam Bazan-Peregrino, Sue Phipps, Sarah Hale, Vivien Mautner, Leonard W. Seymour, Kerry D. Fisher 
Molecular Therapy 
Volume 14, Issue 1, Pages (July 2006)
DOI: /j.ymthe
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Measurement of association of adenovirus particles with blood cells by Q-PCR. (A) Adenovirus particles (Adluc; 3 × 107/ml) were incubated with whole CPD-treated human or murine blood (first two bars) or physiological concentrations of purified human erythrocytes, neutrophils, or PBMC (3.4 × 109, 3.0 × 106, or 2 × 106/ml, respectively) in PBS, FCS (90% vol/vol), or autologous plasma (90% vol/vol) at 37°C. After 30 min the cellular fraction was isolated by centrifugation (660g × 1 min) and washed three times with PBS (12 ml). The number of viral genomes remaining associated with the blood cells was measured by Q-PCR and compared to a standard curve generated using virus particles spiked into blood cell lysates. Results are shown for cells and plasma from three healthy donors, D8 (dotted bars), D6 (black bars), and D4 (open bars). Difference was assessed using unpaired t test; *P < 0.05, ***P < 0.001; †no significance. (B) Blood cell binding of type 5 adenovirus (Adluc) was assessed at three different input doses (7.5 × 106, 7.5 × 107, 7.5 × 108 particles) added to washed human whole blood cells (8.4 × 108 cells) in a total volume of 250 μl. After incubation at 37°C (30 min) cells were isolated by centrifugation (664g × 1 min) and washed twice (12-ml vol of PBS) and virus content was measured by Q-PCR. Results are expressed as the number of virus particles associated with the blood cells (gray bars) and supernatant (black bars). No viruses were added to control cells. Virus recovery was 74.3 ± 31.0, 84.9 ± 11.4, and 121.2 ± 7.4 (mean ± SD) for doses of 7.5 × 106, 7.5 × 107, and 7.5 × 108 particles, respectively. (C) The possible involvement of RGD in binding of adenovirus to human blood cells was assessed in vitro using washed cells (8.4 × 108 cells) in a total volume of 250 μl. Type 5 adenovirus particles (Adluc; 3 × 107 particles/ml) were added in the absence or presence of free RGD peptide (200 μg/ml), and an RGD-deletion mutant type 5 adenovirus (Ad5ΔRGD) was also studied (3 × 107 particles/ml). After incubation at 37°C (30 min) cells and supernatant were isolated by centrifugation (664g × 1 min) and washed twice (12-ml vol of PBS) and virus content was measured by Q-PCR. Results are expressed as the % of virus particles associated with the blood cells (gray bars) and supernatant (black bars).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Inhibition of adenovirus transduction of epithelial cells in the presence of physiological concentrations of human blood cells. (A) Effect of preincubation time on adenovirus infection. Adenovirus particles (Adluc; 3 × 107 particles/ml) were incubated with physiological concentrations of washed human blood cells (3.4 × 109/ml) resuspended in PBS at 37°C for 5–30 min. At each time point 100 μl of the blood cell–virus mixture was added to a monolayer of A549 cells (104 cells/well, seeded 24 h earlier) in a 96-well plate (solid line); simultaneously at each time point blood cells were removed by centrifugation (660g × 1 min) and 100 μl of supernatant was added to A549 cells (dashed line). After a further 90 min the A549 cells were washed and reincubated in fresh infection medium, and luciferase expression was measured after 24 h. The level of luciferase expression achieved without any exposure of virus to blood cells is shown on the y axis. (B) Influence of agitation of blood cells on inhibition of virus infection of epithelial cells. Adenovirus particles (3 × 107 particles/ml) were added to a suspension of whole washed blood cells in PBS (3.4 × 109 cells/ml) and incubated immediately with monolayers of A549 cells in a 96-well plate. The plate was agitated using a Victor II plate reader (Perkin–Elmer, Beaconsfield, UK) at 37°C to prevent settling of blood cells. After 90 min the monolayers were washed and reincubated in fresh infection medium containing 10% FCS. Luciferase expression was measured after 24 h for cells treated with (a) virus alone without agitation, (b) virus and blood cells without agitation, or (c) virus and blood cells with continuous agitation. (C) Lack of effect of murine whole blood on virus infectivity. Virus particles were added to citrate-treated whole murine blood and incubated for 30 min at room temperature. Virus in whole blood was then diluted 1 in 10 with infection medium and applied to A549 cells (104/well) at final concentrations of 103–109 particles/well. After 90 min cells were washed and reincubated in fresh infection medium, and transgene expression was measured after 24 h. (D) Comparison of the ability of blood cells to inhibit infection of A549 cells in monolayer or suspension. Adenovirus particles (3 × 107 particles/ml) were added to a suspension of whole washed blood cells in Eppendorf tubes (3.4 × 109 cells/ml) in Hepes-buffered tissue culture medium and incubated at 37°C for 0 or 30 min prior to addition of a suspension of A549 cells (final concentration 105 cells/ml). The samples were then continually mixed using a rotating wheel at 37°C and transgene expression was measured after 24 h. Levels of luciferase expression are shown in (a) A549 cell suspension with virus alone, (b) A549 cell suspensions added to blood cells and virus that had been preincubated for 30 min, and (c) A549 cell suspensions added to blood cells and virus that had not been preincubated. (E) Influence of concentration of blood cells on inhibition of adenovirus infection of A549 cells. Virus (107 particles/well) were incubated with a monolayer of A549 cells (104/well) for 90 min in the presence of serial dilutions of blood cells, ranging from undiluted (0) to 2000-fold dilution in PBS. Cells were then washed and reincubated in fresh medium, and transgene expression was assessed after 24 h.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Neutralization of infection of adenovirus prebound to A549 cells by blood cells and plasma. Adenovirus (1000 ppc) was incubated with A549 cell monolayers (plated at 104 cells/well in 96-well plates, 24 h earlier) for 1–90 min in the presence of PBS. Cells were then washed and reincubated (without virus) in PBS, CPD-treated whole blood, plasma, or washed blood cells (3.4 × 109 cells/ml) for a further 90 min. The inoculum was removed, cells were washed again and reincubated in infection medium, and luciferase expression was measured after 24 h. Data at time 0 indicate expression levels when virus particles were mixed simultaneously with blood components and exposed to cells for 90 min.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Adenovirus mediates transgene expression in PBMC in the presence of whole human blood. Whole blood from donors D1, D4, and D8 was collected into EDTA tubes and Adluc (109 particles/ml) was added to 1-ml aliquots. After 30 min (37°C) the samples were diluted 1:1 in DMEM and incubated for 24 h on a rotating platform. For analysis of transgene expression, PBMC were purified by Lymphoprep after 24 h and luciferase was quantified in the purified preparation. Results are expressed as light units per milliliter of blood.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Flow cytometry analysis of AdGFP expression in human leukocytes. Human leukocytes (6 × 106 cells/ml) isolated from donor D4 were exposed to AdGFP (100–10,000 ppc) in DMEM, and transgene expression was measured after 14 h. (A) Flow cytometry histograms for different leukocyte populations identified by forward and side scatter. The black line represents virus-treated cells and the gray line represents untreated cells. (B) Flow cytometry analysis of AdGFP expression in gate 2, dual stained with anti-CD14–PE at 100–10,000 ppc as indicated. Control cells, not exposed to virus (0 ppc), are also shown.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Factors influencing Adluc transgene expression in PBMC. (A) Time dependence of transgene expression. PBMC (4 × 105/200 μl/well; black bars) or A549 cells (4 × 104/well; open bars) were exposed to 100 ppc Adluc for 90 min. The cells were then washed twice in PBS and reincubated in fresh medium. At intervals (including the first 90 min incubation, t = 0) the cells were washed and frozen. At the end of the experiment samples from all time points were analyzed for luciferase expression using the Luciferase Assay Kit (Promega). The background level at t = 0 was subtracted from all data points. (B) Time dependence of infection. PBMC (2 × 106/ml) were exposed to 100 ppc Adluc in infection medium for 5–120 min, washed, and resuspended in fresh medium for 24 h before analysis of luciferase activity. The final bar indicates the luciferase expression level for virus left on cells continually without washing. (C) Effect of plasma on transgene expression in A549 cells. A549 cells (4 × 104/well) were incubated with 100 ppc Adluc for 90 min in the presence or absence of 90% plasma from healthy donors D1–D3 and D5–D7, washed, and reincubated and transgene expression was measured after 24 h (black bars). The level of expression achieved in medium alone is shown for comparison (open bar). (D) Effect of autologous plasma on transgene expression in PBMC. PBMC (2 × 106/ml) were exposed to 100 ppc Adluc in tissue culture medium or 90% autologous plasma from the same donors. After 30 min the cells were washed twice in PBS (12 ml) and resuspended in fresh medium; transgene expression was assayed after a further 24 h. For each donor the levels of transgene expression in PBMC are shown following transduction in medium (open bars) or in the presence of autologous plasma (black bars).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Association of adenovirus with blood cells in vivo. (A) A patient with hepatic metastatic colorectal cancer was administered 109 particles of E1-, E3-deleted type 5 adenovirus by direct intratumoral administration under ultrasound guidance, part of a larger clinical study. Samples of venous blood were taken immediately following injection (time 0) and after 0.5, 1, 2, and 4 h and put into citrate vacutainers. Samples were divided in two and part was centrifuged (2000 rpm × 20 min) for preparation of plasma. Virus DNA was extracted and quantified using Q-PCR with primers and probe specific for the CMV promoter–NTR junction, as described elsewhere [5]. The solid line represents virus genomes measured in whole blood, while the dotted line represents virus genomes free in the plasma. (B) Adluc (1011 particles/mouse in 100 μl PBS) was injected iv into Balb/c mice and blood samples (20 μl) were taken from the tail vein (2, 10, 30 min) and immediately diluted with 180 μl PBS to prevent clotting. Cells were separated by centrifugation (2000 rpm × 20 min) and genomes quantified in both cell pellet and supernatant plasma by Q-PCR, using primers and probe specific for the knob–turn region of the fiber gene. The solid line represents virus genomes associated with blood cells, while the dotted line represents virus genomes free in the plasma.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions